G2Cdb::Gene report

Gene id
Gene symbol
Gap43 (MGI)
Mus musculus
growth associated protein 43
G00001491 (Homo sapiens)

Databases (11)

Curated Gene
OTTMUSG00000016275 (Vega mouse gene)
ENSMUSG00000047261 (Ensembl mouse gene)
14432 (Entrez Gene)
65 (G2Cdb plasticity & disease)
Gene Expression
NM_008083 (Allen Brain Atlas)
g01891 (BGEM)
14432 (Genepaint)
gap43 (gensat)
162060 (OMIM)
Marker Symbol
MGI:95639 (MGI)
Protein Sequence
P06837 (UniProt)

Synonyms (4)

  • B-50
  • Basp2
  • GAP-43
  • neuromodulin

Literature (212)

Pubmed - other

  • Peripheral contributions to olfactory bulb cell populations (migrations towards the olfactory bulb).

    Blanchart A, Martín-López E, De Carlos JA and López-Mascaraque L

    Instituto Cajal, CSIC, Department of Cellular, Molecular and Developmental Neurobiology, Madrid, Spain.

    The olfactory system represents one of the most suitable models to study interactions between the peripheral and central nervous systems. The developing olfactory epithelium (olfactory placode and pit) gives rise to several cell populations that migrate towards the telencephalic vesicle. One of these cell populations, called the Migratory Mass (MM), accompanies the first emerging olfactory axons from the olfactory placode, but the fate of these cells and their contribution to the Olfactory Bulb (OB) populations has not been properly addressed. To asses this issue we performed ultrasound-guided in utero retroviral injections at embryonic day (E) 11 revealing the MM as an early source of Olfactory Ensheathing Cells in later postnatal stages. Employing a wide number of antibodies to identify the nature of the infected cells we described that those cells generated within the MM at E11 belong to different cell populations both in the mesenchyma, where they envelop olfactory axons and express the most common glial markers, and in the olfactory bulb, where they are restricted to the Olfactory Nerve and Glomerular layers. Thus, the data reveal the existence of a novel progenitor class within the MM, potentially derived from the olfactory placode which gives rise to different neural cell population including some CNS neurons, glia and olfactory ensheathing cells.

    Glia 2011;59;2;278-92

  • A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

    Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I, Lin-Marq N, Koch M, Bilio M, Cantiello I, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Nürnberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dollé P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G and Ballabio A

    Telethon Institute of Genetics and Medicine, Naples, Italy.

    Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

    Funded by: Medical Research Council: MC_U127527203; Telethon: TGM11S03

    PLoS biology 2011;9;1;e1000582

  • Neuronal transcriptional repressor REST suppresses an Atoh7-independent program for initiating retinal ganglion cell development.

    Mao CA, Tsai WW, Cho JH, Pan P, Barton MC and Klein WH

    Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, 77030, USA. cmao@mdanderson.org

    As neuronal progenitors differentiate into neurons, they acquire a unique set of transcription factors. The transcriptional repressor REST prevents progenitors from undergoing differentiation. Notably, REST binding sites are often associated with retinal ganglion cell (RGC) genes whose expression in the retina is positively controlled by Atoh7, a factor essential for RGC formation. The key regulators that enable a retinal progenitor cell (RPC) to commit to an RGC fate have not been identified. We show here that REST suppresses RGC gene expression in RPCs. REST inactivation causes aberrant expression of RGC transcription factors in proliferating RPCs, independent of Atoh7, resulting in increased RGC formation. Strikingly, inactivating REST in Atoh7-null retinas restores transcription factor expression, which partially activates downstream RGC genes but is insufficient to prevent RGC loss. Our results demonstrate an Atoh7-independent program for initial activation of RGC genes and suggest a novel role for REST in preventing premature expression in RPCs.

    Funded by: NCI NIH HHS: CA016672, P30 CA016672; NEI NIH HHS: EY010608-139005, EY011930, R01 EY011930, R01 EY011930-13; NIGMS NIH HHS: GM081627, P01 GM081627

    Developmental biology 2011;349;1;90-9

  • Hyperpolarization-activated cyclic nucleotide-gated channels in olfactory sensory neurons regulate axon extension and glomerular formation.

    Mobley AS, Miller AM, Araneda RC, Maurer LR, Müller F and Greer CA

    Department of Neurosurgery and Neurobiology, Interdepartmental Neuroscience Program, Yale University School of Medicine, New Haven, Connecticut 06520-8082, USA.

    Mechanisms influencing the development of olfactory bulb glomeruli are poorly understood. While odor receptors (ORs) play an important role in olfactory sensory neuron (OSN) axon targeting/coalescence (Mombaerts et al., 1996; Wang et al., 1998; Feinstein and Mombaerts, 2004), recent work showed that G protein activation alone is sufficient to induce OSN axon coalescence (Imai et al., 2006; Chesler et al., 2007), suggesting an activity-dependent mechanism in glomerular development. Consistent with these data, OSN axon projections and convergence are perturbed in mice deficient for adenylyl cyclase III, which is downstream from the OR and catalyzes the conversion of ATP to cAMP. However, in cyclic nucleotide-gated (CNG) channel knock-out mice OSN axons are only transiently perturbed (Lin et al., 2000), suggesting that the CNG channel may not be the sole target of cAMP. This prompted us to investigate an alternative channel, the hyperpolarization-activated, cyclic nucleotide-gated cation channel (HCN), as a potential developmental target of cAMP in OSNs. Here, we demonstrate that HCN channels are developmentally precocious in OSNs and therefore are plausible candidates for affecting OSN axon development. Inhibition of HCN channels in dissociated OSNs significantly reduced neurite outgrowth. Moreover, in HCN1 knock-out mice the formation of glomeruli was delayed in parallel with perturbations of axon organization in the olfactory nerve. These data support the hypothesis that the outgrowth and coalescence of OSN axons is, at least in part, subject to activity-dependent mechanisms mediated via HCN channels.

    Funded by: NIA NIH HHS: P01 AG028054; NIDCD NIH HHS: F30 DC010324, F32 DC010098, F32 DC010098-01A1, R01 DC000210, R01 DC009817; NIGMS NIH HHS: GM07205, T32 GM007205; NINDS NIH HHS: NS 007224-24, T32 NS007224

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;49;16498-508

  • Axon growth and guidance genes identify nascent, immature, and mature olfactory sensory neurons.

    McIntyre JC, Titlow WB and McClintock TS

    Department of Physiology, University of Kentucky, Lexington, Kentucky 40536-0298, USA.

    Neurogenesis of projection neurons requires that axons be initiated, extended, and connected. Differences in the expression of axon growth and guidance genes must drive these events, but comprehensively characterizing these differences in a single neuronal type has not been accomplished. Guided by a catalog of gene expression in olfactory sensory neurons (OSNs), in situ hybridization and immunohistochemistry revealed that Cxcr4 and Dbn1, two axon initiation genes, marked the developmental transition from basal progenitor cells to immature OSNs in the olfactory epithelium. The CXCR4 immunoreactivity of these nascent OSNs overlapped partially with markers of proliferation of basal progenitor cells and partially with immunoreactivity for GAP43, the canonical marker of immature OSNs. Intracellular guidance cue signaling transcripts Ablim1, Crmp1, Dypsl2, Dpysl3, Dpysl5, Gap43, Marcskl1, and Stmn1-4 were specific to, or much more abundant in, the immature OSN layer. Receptors that mediate axonal inhibition or repulsion tended to be expressed in both immature and mature OSNs (Plxna1, Plxna4, Nrp2, Efna5) or specifically in mature OSNs (Plxna3, Unc5b, Efna3, Epha5, Epha7), although some were specific to immature OSNs (Plxnb1, Plxnb2, Plxdc2, Nrp1). Cell adhesion molecules were expressed either by both immature and mature OSNs (Dscam, Ncam1, Ncam2, Nrxn1) or solely by immature OSNs (Chl1, Nfasc1, Dscaml1). Given the loss of intracellular signaling protein expression, the continued expression of guidance cue receptors in mature OSNs is consistent with a change in the role of these receptors, perhaps to sending signals back to the cell body and nucleus.

    Funded by: NIDCD NIH HHS: R01 DC002736, R01 DC007194

    Journal of neuroscience research 2010;88;15;3243-56

  • Resistance to change and vulnerability to stress: autistic-like features of GAP43-deficient mice.

    Zaccaria KJ, Lagace DC, Eisch AJ and McCasland JS

    Department of Cell & Developmental Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA. zaccarik@upstate.edu

    There is an urgent need for animal models of autism spectrum disorder (ASD) to understand the underlying pathology and facilitate development and testing of new treatments. The synaptic growth-associated protein-43 (GAP43) has recently been identified as an autism candidate gene of interest. Our previous studies show many brain abnormalities in mice lacking one allele for GAP43 [GAP43 (+/-)] that are consistent with the disordered connectivity theory of ASD. Thus, we hypothesized that GAP43 (+/-) mice would show at least some autistic-like behaviors. We found that GAP43 (+/-) mice, relative to wild-type (+/+) littermates, displayed resistance to change, consistent with one of the diagnostic criteria for ASD. GAP43 (+/-) mice also displayed stress-induced behavioral withdrawal and anxiety, as seen in many autistic individuals. In addition, both GAP43 (+/-) mice and (+/+) littermates showed low social approach and lack of preference for social novelty, consistent with another diagnostic criterion for ASD. This low sociability is likely because of the mixed C57BL/6J 129S3/SvImJ background. We conclude that GAP43 deficiency leads to the development of a subset of autistic-like behaviors. As these behaviors occur in a mouse that displays disordered connectivity, we propose that future anatomical and functional studies in this mouse may help uncover underlying mechanisms for these specific behaviors. Strain-specific low sociability may be advantageous in these studies, creating a more autistic-like environment for study of the GAP43-mediated deficits of resistance to change and vulnerability to stress.

    Funded by: NINDS NIH HHS: R01 NS040779, R01NS40779

    Genes, brain, and behavior 2010;9;8;985-96

  • The SRC homology 2 domain protein Shep1 plays an important role in the penetration of olfactory sensory axons into the forebrain.

    Wang L, Vervoort V, Wallez Y, Coré N, Cremer H and Pasquale EB

    Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.

    Shep1 is a multidomain signaling protein that forms a complex with Cas, a key scaffolding component of integrin signaling pathways, to promote the migration of non-neuronal cells. However, the physiological function of Shep1 in the nervous system remains unknown. Interestingly, we found that Shep1 and Cas are both concentrated in the axons of developing olfactory sensory neurons (OSNs). These neurons extend their axons from the olfactory epithelium to the olfactory bulb located at the anterior tip of the forebrain. However, in developing Shep1 knock-out mice, we did not detect penetration of OSN axons across the pial basement membrane surrounding the olfactory bulb, suggesting that Shep1 function is important for the establishment of OSN connections with the olfactory bulb. Interestingly, we observed reduced levels of Cas tyrosine phosphorylation in OSN axons of Shep1 knock-out mice, suggesting compromised Cas signaling function. Indeed, when embedded in a three-dimensional gel of basement membrane proteins, explants from Shep1 knock-out olfactory epithelium extend neuronal processes less efficiently than explants from control epithelium. Furthermore, ectopic expression of Shep1 in non-neuronal cells promotes cell migration through a collagen gel. Later in development, loss of Shep1 function also causes a marked reduction in olfactory bulb size and disruption of bulb lamination, which may be primarily attributable to the defective innervation. The greatly reduced OSN connections and hypoplasia of the olfactory bulb, likely resulting in anosmia, are reminiscent of the symptoms of Kallmann syndrome, a human developmental disease that can be caused by mutations in a growing number of genes.

    Funded by: NCI NIH HHS: CA102583, P01 CA102583, P01 CA102583-05; NICHD NIH HHS: HD025938, P01 HD025938, P01 HD025938-190008

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;39;13201-10

  • Development of innervation to maxillary whiskers in mice.

    Maklad A, Conway M, Hodges C and Hansen LA

    Department of Anatomy, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA. amaklad@umc.edu

    The maxillary vibrissal pad is a unique, richly innervated sensory apparatus. It is highly evolved in the rodent that it constitutes a major source of sensory information to the somatosensory cortex. In this report, indocarbocyanine tracing and immunofluorescence were used to study the embryonic and early neonatal development of innervation to maxillary vibrissal follicles in mice. The first sign of vibrissal follicle innervation occurred at embryonic day 12 (E12), when the lateral nasal and maxillary processes were penetrated by nerve branches with small terminal plexuses assuming the positions of vibrissal follicle primordia. Between E13 and E15, the nerve plexuses at the presumptive follicles grew in size and became more numerous with no signs of specific receptor subtype formation. By E17, the nerve plexuses had grown further in size and the region-specific receptor subtype specification developed. At birth (P0), the superficial vibrissal nerves began to innervate the apical part of the inner conical body, whereas the deep vibrissal nerve gave off the recurrent cavernous branches. At P3, all of the different sets of receptor subtypes had regional distributions, densities and morphologies comparable to those described in adult mice. A 3-day old mouse had all complements of sensory receptors necessary for somatosensory transduction as revealed not only by neuroanatomic tracing but also with immunofluorescence for several markers of neurosensory differentiation. Our data reveal a hitherto unknown time table for the development of peripheral sensory receptors in the vibrissal follicles. This time table parallels that of their central targets in the somatosensory barrel cortex, which develops at P4.

    Anatomical record (Hoboken, N.J. : 2007) 2010;293;9;1553-67

  • Axon fasciculation in the developing olfactory nerve.

    Miller AM, Maurer LR, Zou DJ, Firestein S and Greer CA

    Department of Neurosurgery, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.

    Olfactory sensory neuron (OSN) axons exit the olfactory epithelium (OE) and extend toward the olfactory bulb (OB) where they coalesce into glomeruli. Each OSN expresses only 1 of approximately 1,200 odor receptors (ORs). OSNs expressing the same OR are distributed in restricted zones of the OE. However, within a zone, the OSNs expressing a specific OR are not contiguous - distribution appears stochastic. Upon reaching the OB the OSN axons expressing the same OR reproducibly coalesce into two to three glomeruli. While ORs appear necessary for appropriate convergence of axons, a variety of adhesion associated molecules and activity-dependent mechanisms are also implicated. Recent data suggest pre-target OSN axon sorting may influence glomerular convergence. Here, using regional and OR-specific markers, we addressed the spatio-temporal properties associated with the onset of homotypic fasciculation in embryonic mice and assessed the degree to which subpopulations of axons remain segregated as they extend toward the nascent OB. We show that immediately upon crossing the basal lamina, axons uniformly turn sharply, usually at an approximately 90° angle toward the OB. Molecularly defined subpopulations of axons show evidence of spatial segregation within the nascent nerve by embryonic day 12, within 48 hours of the first OSN axons crossing the basal lamina, but at least 72 hours before synapse formation in the developing OB. Homotypic fasciculation of OSN axons expressing the same OR appears to be a hierarchical process. While regional segregation occurs in the mesenchyme, the final convergence of OR-specific subpopulations does not occur until the axons reach the inner nerve layer of the OB.

    Funded by: NIA NIH HHS: AG028054; NIDCD NIH HHS: DC00210, DC007880, F30 DC010324, R01 DC000210, R01 DC007880; NIGMS NIH HHS: GM0720

    Neural development 2010;5;20

  • Comparative gene expression analysis of genital tubercle development reveals a putative appendicular Wnt7 network for the epidermal differentiation.

    Chiu HS, Szucsik JC, Georgas KM, Jones JL, Rumballe BA, Tang D, Grimmond SM, Lewis AG, Aronow BJ, Lessard JL and Little MH

    Institute for Molecular Bioscience, The University of Queensland, St. Lucia 4072, Australia.

    Here we describe the first detailed catalog of gene expression in the developing lower urinary tract (LUT), including epithelial and mesenchymal portions of the developing bladder, urogenital sinus, urethra, and genital tubercle (GT) at E13 and E14. Top compartment-specific genes implicated by the microarray data were validated using whole-mount in situ hybridization (ISH) over the entire LUT. To demonstrate the potential of this resource to implicate developmentally critical features, we focused on gene expression patterns and pathways in the sexually indeterminate, androgen-independent GT. GT expression patterns reinforced the proposed similarities between development of GT, limb, and craniofacial prominences. Comparison of spatial expression patterns predicted a network of Wnt7a-associated GT-enriched epithelial genes, including Gjb2, Dsc3, Krt5, and Sostdc1. Known from other contexts, these genes are associated with normal epidermal differentiation, with disruptions in Dsc3 and Gjb2 showing palmo-plantar keratoderma in the limb. We propose that this gene network contributes to normal foreskin, scrotum, and labial development. As several of these genes are known to be regulated by, or contain cis elements responsive to retinoic acid, estrogen, or androgen, this implicates this pathway in the later androgen-dependent development of the GT.

    Funded by: NIDDK NIH HHS: DK070136, DK070219, T35 DK060444, U01 DK070136, U01 DK070136-05S1, U01 DK070219

    Developmental biology 2010;344;2;1071-87

  • Postsynaptic deregulation in GAP-43 heterozygous mouse barrel cortex.

    Kelly EA, Tremblay ME, McCasland JS and Majewska AK

    Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.

    Formation of whisker-related barrels in primary somatosensory cortex (S1) requires communication between presynaptic thalamocortical afferents (TCAs) and postsynaptic cortical neurons. GAP-43 is crucially involved in targeting TCAs to postsynaptic S1 neurons but its influence on the interactions between these 2 elements has not been explored. Here, we tested the hypothesis that reduced early expression of presynaptic GAP-43 (GAP-43 heterozygous [HZ] mice) alters postsynaptic differentiation of barrel cells. We found a transient increase in cytochrome oxidase staining between P6 and P14 in HZ animals, indicative of increased metabolic activity in barrel cortex during this time. Golgi impregnation and microtubule-associated protein 2 immunohistochemistry showed anomalous dendritic patterning in GAP-43 HZ cortex at P5, with altered dendritic length and branching and abnormal retention of dendrites that extend into developing septa. This deficiency was no longer apparent at P7, suggesting partial recovery of dendritic pruning processes. Finally, we showed early defects in synaptogenesis from P4 to P5 with increased colocalization of NR1 and GluR1 staining in HZ mice. By P7, this colocalization had normalized to wild type levels. Taken together, our findings suggest abnormal postsynaptic differentiation in GAP-43 HZ cortex during early barrel development, followed by adaptive compensation and partial phenotypic rescue.

    Funded by: NEI NIH HHS: EY019277, R01 EY019277; NINDS NIH HHS: NS31829, NS40779

    Cerebral cortex (New York, N.Y. : 1991) 2010;20;7;1696-707

  • Sequential arrival and graded secretion of Sema3F by olfactory neuron axons specify map topography at the bulb.

    Takeuchi H, Inokuchi K, Aoki M, Suto F, Tsuboi A, Matsuda I, Suzuki M, Aiba A, Serizawa S, Yoshihara Y, Fujisawa H and Sakano H

    Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan.

    In the mouse olfactory system, the anatomical locations of olfactory sensory neurons (OSNs) roughly correlate with their axonal projection sites along the dorsal-ventral (D-V) axis of the olfactory bulb (OB). Here we report that an axon guidance receptor, Neuropilin-2 (Nrp2), and its repulsive ligand, Semaphorin-3F (Sema3F), are expressed by OSNs in a complementary manner that is important for establishing olfactory map topography. Sema3F is secreted by early-arriving axons of OSNs and is deposited at the anterodorsal OB to repel Nrp2-positive axons that arrive later. Sequential arrival of OSN axons as well as the graded and complementary expression of Nrp2 and Sema3F by OSNs help to form the topographic order along the D-V axis.

    Cell 2010;141;6;1056-67

  • MBD2 and MeCP2 regulate distinct transitions in the stage-specific differentiation of olfactory receptor neurons.

    Macdonald JL, Verster A, Berndt A and Roskams AJ

    Department of Zoology, Life Sciences Institute and Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.

    DNA methylation-dependent gene silencing is initiated by DNA methyltransferases (DNMTs) and mediated by methyl-binding domain proteins (MBDs), which recruit histone deacetylases (HDACs) to silence DNA, a process that is essential for normal development. Here, we show that the MBD proteins MBD2 and MeCP2 regulate distinct transitional stages of olfactory receptor neuron (ORN) differentiation in vivo. Mbd2 null progenitors display enhanced proliferation, recapitulated by HDAC inhibition, and Mbd2 null ORNs have a decreased lifespan. Mecp2 null ORNs, on the other hand, temporarily stall at the stage of terminal differentiation, retaining expression of the immature neuronal protein GAP43 after initiating expression of mature neuronal genes. The Gap43 promoter is highly methylated in the mature, but not embryonic olfactory epithelium (OE), suggesting that Gap43 may be regulated by DNA methylation during ORN differentiation. Thus, MBD2 and MeCP2 may mediate distinct, sequential transitions of ORN differentiation-an epigenetic mechanism that may be relevant to developmental regulation throughout the nervous system.

    Funded by: Canadian Institutes of Health Research

    Molecular and cellular neurosciences 2010;44;1;55-67

  • Taste cell formation does not require gustatory and somatosensory innervation.

    Ito A, Nosrat IV and Nosrat CA

    Department of Bioscience Research, College of Dentistry, and Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN, USA.

    Dependency of taste buds and taste papillae on innervation has been debated for a long time. Previous research showed neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), play an important role for the establishment of the lingual gustatory and somatosensory innervation. BDNF null mutant mice showed severe deficits in gustatory innervation and loss of taste buds while NT-3 null mutation reduced lingual somatosensory innervation to tongue papillae. These results proved BDNF or NT-3 null mutations affected different sensory modalities (i.e. gustatory and somatosensory, respectively). In this study, we analyzed taste bud development in BDNFxNT-3 double knockout mice to examine the relationship between taste bud development and gustatory/somatosensory innervation. Our results demonstrate that, at the initial stage, before nerve fibers reached the appropriate areas in the papilla, taste bud formation did not require innervation. However, at the synaptogenic stage, after nerve fibers ramified into the apical epithelium, innervation was required and played an essential role in the development of taste buds/papillae.

    Funded by: NIDCD NIH HHS: R01 DC007628, R01 DC007628-05S1, R01 DC007628-06; PHS HHS: R01-RDC007628

    Neuroscience letters 2010;471;3;189-94

  • Kappa opioid receptor contributes to EGF-stimulated neurite extension in development.

    Tsai NP, Tsui YC, Pintar JE, Loh HH and Wei LN

    Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA.

    Epidermal growth factor (EGF), a mitogen, also stimulates neurite extension during development, but the underlying mechanism is elusive. This study reveals a functional role for kappa opioid receptor (KOR) in EGF-stimulated neurite extension, and the underlying mechanism. EGF and activated EGF receptor (EGFR) levels are elevated in embryonic spinal cords during late gestation stages, with concurrent rise in protein levels of KOR and axon extension markers, growth-associated protein 43 (GAP43), and transient axonal glycoprotein-1 (TAG-1). Both GAP43 and TAG-1 levels are significantly lower in KOR-null (KOR(-/-)) spinal cords, and EGFR inhibitors effectively reduce the levels of KOR, GAP43, and TAG-1 in wild-type embryonic spinal cords. For KOR(-/-) or KOR-knockdown dorsal root ganglion (DRG) neurons, EGF can no longer effectively stimulate axon extension, which can be rescued by introducing a constitutive KOR expressing vector but not by a regulated KOR vector carrying its 5' untranslated region, which can be bound and repressed by growth factor receptor-bound protein 7 (Grb7). Furthermore, blocking KOR activation by application of anti-dynorphin, KOR antagonist, or EGFR inhibitor effectively reduces axon extension of DRG neurons. Thus, EGF-stimulated axon extension during development is mediated, at least partially, by specific elevation of KOR protein production at posttranscriptional level, as well as activation of KOR signaling. The result also reveals an action of EGF to augment posttranscriptional regulation of certain mRNAs during developmental stages.

    Funded by: NIDA NIH HHS: DA11190, DA11806, K02 DA013926, K02-DA13926, P50 DA011806, R01 DA011190; NIDDK NIH HHS: DK54733, DK60521, R01 DK054733, R01 DK060521

    Proceedings of the National Academy of Sciences of the United States of America 2010;107;7;3216-21

  • Ectopic growth of hippocampal mossy fibers in a mutated GAP-43 transgenic mouse with impaired spatial memory retention.

    Holahan MR, Honegger KS and Routtenberg A

    Department of Psychology, Institute of Neuroscience, Carleton University, Ottawa, ON K1S 5B6, Canada. matthew_holahan@carleton.ca

    In a previous study, it was shown that transgenic mice, designated G-NonP, forget the location of a water maze hidden platform when tested 7 days after the last training day (Holahan and Routtenberg (2008) Hippocampus 18:1099-1102). The memory loss in G-NonP mice might be related to altered hippocampal architecture suggested by the fact that in the rat, 7 days after water maze training, there is discernible mossy fiber (MF) growth (Holahan et al. (2006) Hippocampus 16:560-570; Rekart et al. (2007) Learn Mem 14:416-421). In the present report, we studied the distribution of the MF system within the hippocampus of naïve, untrained, G-NonP mouse. In WT mice, the MF projection was restricted to the stratum lucidum of CA3 with no detectable MF innervation in distal stratum oriens (dSO). In G-NonP mice, in contrast, there was an ectopic projection terminating in the CA3 dSO. Unexpectedly, there was nearly a complete loss of immunostaining for the axonal marker Tau1 in the G-NonP transgenic mice in the MF terminal fields indicating that transgenesis itself leads to off-target consequences (Routtenberg (1996) Trends Neurosci 19:471-472). Because transgenic mice overexpressing nonmutated, wild type GAP-43 do not show this ectopic growth (Rekart et al., in press) and the G-NonP mice overexpress a mutated form of GAP-43 precluding its phosphorylation by protein kinase C (PKC), the possibility exists that permanently dephosphorylated GAP-43 disrupts normal axonal fasciculation which gives rise to the ectopic growth into dSO.

    Funded by: NIA NIH HHS: AG20506, T32 AG020506; NIMH NIH HHS: MH65436, R01 MH065436, R01 MH065436-05

    Hippocampus 2010;20;1;58-64

  • Overexpression of GAP-43 reveals unexpected properties of hippocampal mossy fibers.

    Rekart JL and Routtenberg A

    Department of Psychology, Northwestern University, Evanston, IL 60208 USA. aryeh@northwestern.edu

    The mossy fiber (MF) system targets the apical dendrites of CA3 pyramidal cells in the stratum lucidum (SL). In mice overexpressing the growth-associated protein GAP-43 there is an apparent ectopic growth of these MFs into the stratum oriens (SO) targeting the basal dendrites of these same pyramidal cells (Aigner et al. (1995) Cell 83:269-278). This is the first evidence to our knowledge that links increased GAP-43 expression with growth of central axons. Here we studied the Aigner et al. transgenic mice but were unable to confirm such growth into SO. However, using quantitative methods we did observe enhanced growth within the regions normally targeted by MFs, for example, the SL in the CA3a region. These contrasting results led us to study MFs with double-immunostaining using an immunohistochemical marker for MFs, the zinc transporter, ZnT3, to visualize the colocalization of transgenic GAP-43 within MFs. Unexpectedly, using both fluorescence and confocal microscopy, we were unable to detect colocalization of GAP-43-positive axons with ZnT3-positive MF axons within the MF pathways, either in the region of the MF axons or in the SL, where MF terminals are abundant. In contrast, the plasma membrane-associated presynaptic marker SNAP-25 did colocalize with transgenic GAP-43-positive terminals in the SL. Synaptophysin, the vesicle-associated presynaptic terminal marker, colocalized with ZnT3 but did not appear to colocalize with GAP-43. The present findings raise important questions about the properties of granule cells and the MF mechanisms that differentially regulate axonal remodeling in the adult hippocampus: (1) Because there appears to be at least two populations of granule cells defined by their differential protein expression, this points to the existence of an intrinsic heterogeneity of granule cell expression beyond that contributed by adult neurogenesis; (2) Giventhe present evidence that growth is induced in mice overexpressing GAP-43 in adjacent non-GAP-43 containing MFs, the potential exists for a heretofore unexplored interaxonal communication mechanism.

    Funded by: NIMH NIH HHS: MH TG 067564, MH65436-06, R01 MH065436, R01 MH065436-06A2, T32 MH067564, T32 MH067564-06

    Hippocampus 2010;20;1;46-57

  • Multiple non-cell-autonomous defects underlie neocortical callosal dysgenesis in Nfib-deficient mice.

    Piper M, Moldrich RX, Lindwall C, Little E, Barry G, Mason S, Sunn N, Kurniawan ND, Gronostajski RM and Richards LJ

    Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia. m.piper@uq.edu.au

    Background: Agenesis of the corpus callosum is associated with many human developmental syndromes. Key mechanisms regulating callosal formation include the guidance of axons arising from pioneering neurons in the cingulate cortex and the development of cortical midline glial populations, but their molecular regulation remains poorly characterised. Recent data have shown that mice lacking the transcription factor Nfib exhibit callosal agenesis, yet neocortical callosal neurons express only low levels of Nfib. Therefore, we investigate here how Nfib functions to regulate non-cell-autonomous mechanisms of callosal formation.

    Results: Our investigations confirmed a reduction in glial cells at the midline in Nfib-/- mice. To determine how this occurs, we examined radial progenitors at the cortical midline and found that they were specified correctly in Nfib mutant mice, but did not differentiate into mature glia. Cellular proliferation and apoptosis occurred normally at the midline of Nfib mutant mice, indicating that the decrease in midline glia observed was due to deficits in differentiation rather than proliferation or apoptosis. Next we investigated the development of callosal pioneering axons in Nfib-/- mice. Using retrograde tracer labelling, we found that Nfib is expressed in cingulate neurons and hence may regulate their development. In Nfib-/- mice, neuropilin 1-positive axons fail to cross the midline and expression of neuropilin 1 is diminished. Tract tracing and immunohistochemistry further revealed that, in late gestation, a minor population of neocortical axons does cross the midline in Nfib mutants on a C57Bl/6J background, forming a rudimentary corpus callosum. Finally, the development of other forebrain commissures in Nfib-deficient mice is also aberrant.

    Conclusion: The formation of the corpus callosum is severely delayed in the absence of Nfib, despite Nfib not being highly expressed in neocortical callosal neurons. Our results indicate that in addition to regulating the development of midline glial populations, Nfib also regulates the expression of neuropilin 1 within the cingulate cortex. Collectively, these data indicate that defects in midline glia and cingulate cortex neurons are associated with the callosal dysgenesis seen in Nfib-deficient mice, and provide insight into how the development of these cellular populations is controlled at a molecular level.

    Neural development 2009;4;43

  • Role of interleukin-15 in the development of mouse olfactory nerve.

    Umehara T, Udagawa J, Takamura K, Kimura M, Ishimitsu R, Kiyono H, Kawauchi H and Otani H

    Department of Otorhinolaryngology, Faculty of Medicine, Shimane University, Izumo, Japan. umehara@med.shimane-u.ac.jp

    Interleukin (IL)-15 interacts with components of the IL-2 receptor (R) and exhibits T cell-stimulating activity similar to that of IL-2. In addition, IL-15 is widely expressed in many cell types and tissues, including the central nervous system. We provide evidence of a novel role of IL-15 in olfactory neurogenesis. Both IL-15 and IL-15R alpha were expressed in neuronal precursor cells of the developing olfactory epithelium in mice. Adult IL-15R alpha knockout mice had fewer mature olfactory neurons and proliferating cells than wild-type. Our results suggest that IL-15 plays an important role in regulating cell proliferation in olfactory neurogenesis.

    Congenital anomalies 2009;49;4;253-7

  • Overexpression of GAP-43 modifies the distribution of the receptors for myelin-associated growth-inhibitory proteins in injured Purkinje axons.

    Foscarin S, Gianola S, Carulli D, Fazzari P, Mi S, Tamagnone L and Rossi F

    Department of Neuroscience, Neuroscience Institute of Turin (NIT), University of Turin, Turin, Italy.

    Abstract Neurons with enhanced intrinsic growth capabilities can elongate their axons into non-permissive territories, but the mechanisms that enable the outgrowing processes to overcome environmental inhibition are largely unknown. To address this issue, we examined adult mouse Purkinje cells that overexpress the axonal growth-associated protein GAP-43. After injury, these neurons exhibit sprouting along the intracortical neuritic course and at the severed stump in the white matter. To determine whether GAP-43-overexpressing Purkinje cells are responsive to extrinsic inhibitory cues, we investigated the content and subcellular localization of major receptors for myelin-associated inhibitory proteins, PlexinB1 and the Nogo receptor (NgR) with the related co-receptors LINGO-1 and p75. Expression of these molecules, estimated by measuring perikaryal immunostaining intensity and Western blot, was not different in wild-type or transgenic mice, and it was not overtly modified after axotomy. Following injury, however, the content of PlexinB1 was significantly reduced in GAP-43-overexpressing neurites. Furthermore, in the same axons the distribution of both PlexinB1 and NgR was altered, being inverse to that of GAP-43. Labelling for the two receptors was conspicuously reduced on the axonal surface and it was almost undetectable in the outgrowing sprouts, which showed strong GAP-43 immunoreactivity. These observations indicate that although GAP-43 overexpression does not modify the expression of receptors for myelin-associated inhibitory factors, it interferes with their subcellular localization and exposure on the neuritic membrane. Therefore, GAP-43 promotes axon growth by multiple synergistic mechanisms that potentiate the intrinsic motility of the elongating processes, while reducing their sensitivity to environmental inhibition.

    The European journal of neuroscience 2009;30;10;1837-48

  • Expression of the neural specific protein, GAP-43, dramatically lengthens the cell cycle in fibroblasts.

    Zhao J, Yao Y, Xu C, Jiang X and Xu Q

    Beijing Institute for Neuroscience, Beijing Center for Neural Regeneration and Repair, Key Laboratory for Neurodegenerative Diseases of the Ministry of Education, Capital Medical University, Beijing 100069, PR China.

    It has been demonstrated that during neurogenesis in the mammalian brain, cell-cycle lengthening in neuronal progenitors may cause them to switch from proliferation to neuron-generating division. However, little is known about the cellular mechanisms involved in lengthening of the cell cycle. Growth-associated protein-43 (GAP-43) is a nervous system-specific protein whose expression in proliferating neuroblasts is related to neurogenesis. In this study, we investigated the effect of GAP-43 on cell-cycle progression in transgenic fibroblast cells. Using cumulative bromodeoxyuridine labeling, cell-cycle kinetics in GAP-43-transgenic and control NIH 3T3 cells were analyzed. Our data demonstrate that expression of GAP-43 in fibroblasts results in lengthening of the cell cycle compared to control fibroblasts. The mechanism by which GAP-43 mediated this effect appeared to involve increasing the time spent by the cells in the G(1) phase of the cell cycle.

    International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience 2009;27;6;531-7

  • The N-Myc-DLL3 cascade is suppressed by the ubiquitin ligase Huwe1 to inhibit proliferation and promote neurogenesis in the developing brain.

    Zhao X, D' Arca D, Lim WK, Brahmachary M, Carro MS, Ludwig T, Cardo CC, Guillemot F, Aldape K, Califano A, Iavarone A and Lasorella A

    Institute for Cancer Genetics, Columbia University Medical Center, New York, NY 10032, USA.

    Self-renewal and proliferation of neural stem cells and the decision to initiate neurogenesis are crucial events directing brain development. Here we show that the ubiquitin ligase Huwe1 operates upstream of the N-Myc-DLL3-Notch pathway to control neural stem cell activity and promote neurogenesis. Conditional inactivation of the Huwe1 gene in the mouse brain caused neonatal lethality associated with disorganization of the laminar patterning of the cortex. These defects stemmed from severe impairment of neurogenesis associated with uncontrolled expansion of the neural stem cell compartment. Loss- and gain-of-function experiments in the mouse cortex demonstrated that Huwe1 restrains proliferation and enables neuronal differentiation by suppressing the N-Myc-DLL3 cascade. Notably, human high-grade gliomas carry focal hemizygous deletions of the X-linked Huwe1 gene in association with amplification of the N-myc locus. Our results indicate that Huwe1 balances proliferation and neurogenesis in the developing brain and that this pathway is subverted in malignant brain tumors.

    Funded by: Medical Research Council: MC_U117570528; NCI NIH HHS: R01 CA085628, R01 CA109755, R01 CA131126, R01 CA131126-01A1, R01CA085628, R01CA109755, R01CA131126, U54 CA121852, U54CA121852

    Developmental cell 2009;17;2;210-21

  • Dual acylation is required for trafficking of growth-associated protein-43 (GAP-43) to endosomal recycling compartment via an Arf6-associated endocytic vesicular pathway.

    Trenchi A, Gomez GA and Daniotti JL

    Centro de Investigaciones en Química Biológica de Córdoba (UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba X5000HUA, Argentina.

    GAP-43 (growth-associated protein-43) is a dually palmitoylated protein, at cysteine residues at positions 3 and 4, that mostly localizes in plasma membrane both in neural and non-neural cells. In the present study, we have examined membrane association, subcellular distribution and intracellular trafficking of GAP-43 in CHO (Chinese hamster ovary)-K1 cells. Using biochemical assays and confocal and video microscopy in living cells we demonstrated that GAP-43, at steady state, localizes at the recycling endosome in addition to the cytoplasmic leaflet of the plasma membrane and TGN (trans-Golgi network). Pharmacological inhibition of newly synthesized GAP-43 acylation or double mutation of Cys3 and Cys4 of GAP-43 completely disrupts TGN, plasma membrane and recycling endosome association. A combination of selective photobleaching techniques and time-lapse fluorescence microscopy reveals a dynamic association of GAP-43 with recycling endosomes in equilibrium with the plasma membrane pool. Newly synthesized GAP-43 is found mainly associated with the TGN, but not with the pericentriolar recycling endosome, and traffics to the plasma membrane by a brefeldin A-insensitive pathway. Impairment of plasma membrane fusion and internalization by treatment with tannic acid does affect the trafficking of GAP-43 from plasma membrane to recycling endosomes which reveals a vesicle-mediated retrograde trafficking of GAP-43. Here, we also show that internalization of GAP-43 is regulated by Arf (ADP-ribosylation factor) 6. Taken together, these results demonstrate that dual acylation is required for sorting of peripheral membrane-associated GAP-43 to recycling endosome via an Arf6-associated endocytic vesicular pathway.

    The Biochemical journal 2009;421;3;357-69

  • NFAT-3 is a transcriptional repressor of the growth-associated protein 43 during neuronal maturation.

    Nguyen T, Lindner R, Tedeschi A, Forsberg K, Green A, Wuttke A, Gaub P and Di Giovanni S

    Laboratory for NeuroRegeneration and Repair, Department of Neurology, Hertie Institute for Clinical Brain Research, Germany.

    Transcription is essential for neurite and axon outgrowth during development. Recent work points to the involvement of nuclear factor of activated T cells (NFAT) in the regulation of genes important for axon growth and guidance. However, NFAT has not been reported to directly control the transcription of axon outgrowth-related genes. To identify transcriptional targets, we performed an in silico promoter analysis and found a putative NFAT site within the GAP-43 promoter. Using in vitro and in vivo experiments, we demonstrated that NFAT-3 regulates GAP-43, but unexpectedly, does not promote but represses the expression of GAP-43 in neurons and in the developing brain. Specifically, in neuron-like PC-12 cells and in cultured cortical neurons, the overexpression of NFAT-3 represses GAP-43 activation mediated by neurotrophin signaling. Using chromatin immunoprecipitation assays, we also show that prior to neurotrophin activation, endogenous NFAT-3 occupies the GAP-43 promoter in PC-12 cells, in cultured neurons, and in the mouse brain. Finally, we observe that NFAT-3 is required to repress the physiological expression of GAP-43 and other pro-axon outgrowth genes in specific developmental windows in the mouse brain. Taken together, our data reveal an unexpected role for NFAT-3 as a direct transcriptional repressor of GAP-43 expression and suggest a more general role for NFAT-3 in the control of the neuronal outgrowth program.

    Funded by: NINDS NIH HHS: R21 NS052640

    The Journal of biological chemistry 2009;284;28;18816-23

  • Protein kinase C-dependent trafficking of glutamate transporters excitatory amino acid carrier 1 and glutamate transporter 1b in cultured cerebellar granule cells.

    Karatas-Wulf U, Koepsell H, Bergert M, Sönnekes S and Kugler P

    University of Würzburg, Institute of Anatomy and Cell Biology, Würzburg, Germany.

    Previous data showed that cell surface expression of the glutamate transporters GLT1a and excitatory amino acid carrier 1 (EAAC1), localized in glia and neurons of the CNS, can be regulated by protein kinase C (PKC). Regulation and physiological importance of GLT1b, a splice variant of GLT1a, is not understood. In the present study we used cultured cerebellar granule cells (CGCs) from mice to investigate PKC dependent trafficking of GLT1b in comparison to GLT1a and EAAC1 using immunohistochemistry and subcellular fractionation followed by Western blotting. In neurites of CGCs, GLT1b and EAAC1 were localized to different aggregates of vesicles that were different from vesicle aggregates containing vesicular glutamate transporters. In CGCs cultured with low-potassium medium, stimulation of PKC by phorbol ester enhanced the formation of large varicosities in neurites that exhibited immunoreactivity for GLT1a, GLT1b, and EAAC1. Stimulation of PKC leads to a significant increase of GLT1b and EAAC1 in the plasma membrane whereas GLT1a in the plasma membrane was decreased. Following PKC stimulation, also a significant increase of transporter-mediated glutamate uptake representing sodium dependent glutamate uptake, was observed. Similarly, the fraction of glutamate uptake, that was sensitive to the inhibitor WAY-213613 and represents uptake by GLT1a and GLT1b, was increased after stimulation by PKC. The findings suggest that PKC is similarly involved in regulation of surface trafficking of GLT1b and EAAC1 and that PKC stimulated increase in surface location of GLT1b and EAAC1 in glutamatergic CGCs.

    Neuroscience 2009;161;3;794-805

  • The level of the transcription factor Pax6 is essential for controlling the balance between neural stem cell self-renewal and neurogenesis.

    Sansom SN, Griffiths DS, Faedo A, Kleinjan DJ, Ruan Y, Smith J, van Heyningen V, Rubenstein JL and Livesey FJ

    Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.

    Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. We used Pax6 as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. We identified the genomic binding locations of Pax6 in neocortical stem cells during normal development and ascertained the functional significance of genes that we found to be regulated by Pax6, finding that Pax6 positively and directly regulates cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, we defined a core network regulating neocortical stem cell decision-making in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. We find that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal.

    Funded by: Cancer Research UK; Medical Research Council: MC_U127527199; NINDS NIH HHS: R01 NS034661, R01 NS099099; Wellcome Trust

    PLoS genetics 2009;5;6;e1000511

  • Fate mapping of mammalian embryonic taste bud progenitors.

    Thirumangalathu S, Harlow DE, Driskell AL, Krimm RF and Barlow LA

    Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO 80045, USA.

    Mammalian taste buds have properties of both epithelial and neuronal cells, and are thus developmentally intriguing. Taste buds differentiate at birth within epithelial appendages, termed taste papillae, which arise at mid-gestation as epithelial thickenings or placodes. However, the embryonic relationship between placodes, papillae and adult taste buds has not been defined. Here, using an inducible Cre-lox fate mapping approach with the ShhcreER(T2) mouse line, we demonstrate that Shh-expressing embryonic taste placodes are taste bud progenitors, which give rise to at least two different adult taste cell types, but do not contribute to taste papillae. Strikingly, placodally descendant taste cells disappear early in adult life. As placodally derived taste cells are lost, we used Wnt1Cre mice to show that the neural crest does not supply cells to taste buds, either embryonically or postnatally, thus ruling out a mesenchymal contribution to taste buds. Finally, using Bdnf null mice, which lose neurons that innervate taste buds, we demonstrate that Shh-expressing taste bud progenitors are specified and produce differentiated taste cells normally, in the absence of gustatory nerve contact. This resolution of a direct relationship between embryonic taste placodes with adult taste buds, which is independent of mesenchymal contribution and nerve contact, allows us to better define the early development of this important sensory system. These studies further suggest that mammalian taste bud development is very distinct from that of other epithelial appendages.

    Funded by: NIDCD NIH HHS: DC007176, DC04657, DC08373, P30 DC004657, R01 DC008373

    Development (Cambridge, England) 2009;136;9;1519-28

  • A p53-CBP/p300 transcription module is required for GAP-43 expression, axon outgrowth, and regeneration.

    Tedeschi A, Nguyen T, Puttagunta R, Gaub P and Di Giovanni S

    Laboratory for NeuroRegeneration and Repair, Department of Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, Otfried-Mueller Strasse 27, Tuebingen, Germany.

    Transcription regulates axon outgrowth and regeneration. However, to date, no transcription complexes have been shown to control axon outgrowth and regeneration by regulating axon growth genes. Here, we report that the tumor suppressor p53 and its acetyltransferases CBP/p300 form a transcriptional complex that regulates the axonal growth-associated protein 43, a well-characterized pro-axon outgrowth and regeneration protein. Acetylated p53 at K372-3-82 drives axon outgrowth, GAP-43 expression, and binds specific elements on the neuronal GAP-43 promoter in a chromatin environment through CBP/p300 signaling. Importantly, in an axon regeneration model, both CBP and p53 K372-3-82 are induced following axotomy in facial motor neurons, where p53 K372-3-82 occupancy of GAP-43 promoter is enhanced as shown by in vivo chromatin immunoprecipitation. Finally, by comparing wild-type and p53 null mice, we demonstrate that the p53/GAP-43 transcriptional module is specifically switched on during axon regeneration in vivo. These data contribute to the understanding of gene regulation in axon outgrowth and may suggest new molecular targets for axon regeneration.

    Funded by: NINDS NIH HHS: R21 NS 052640

    Cell death and differentiation 2009;16;4;543-54

  • GAP-43 is essential for the neurotrophic effects of BDNF and positive AMPA receptor modulator S18986.

    Gupta SK, Mishra R, Kusum S, Spedding M, Meiri KF, Gressens P and Mani S

    National Brain Research Center, Manesar, Haryana, India.

    Positive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor modulators include benzamide compounds that allosterically modulate AMPA glutamate receptors. These small molecules that cross the blood-brain barrier have been shown to act as a neuroprotectant by increasing the levels of endogenous brain-derived neurotrophic factor (BDNF). Positive AMPA receptor modulators have also been shown to increase the levels of growth-associated protein-43 (GAP-43). GAP-43 plays a major role in many aspects of neuronal function in vertebrates. The goal of this study was to determine whether GAP-43 was important in mediating the actions of positive AMPA receptor modulator (S18986) and BDNF. Using cortical cultures from GAP-43 knockout and control mice, we show that (1) GAP-43 is upregulated in response to S18986 and BDNF in control cultures; (2) this upregulation of GAP-43 is essential for mediating the neuroprotective effects of S18986 and BDNF; (3) administration of S18986 and BDNF leads to an increase in the expression of the glutamate transporters GLT-1 and GLAST that are key to limiting excitotoxic cell death and this increase in GLT-1 and GLAST expression is completely blocked in the absence of GAP-43. Taken together this study concludes that GAP-43 is an important mediator of the neurotrophic effects of S18986 and BDNF on neuronal survival and plasticity, and is essential for the success of positive AMPA receptor modulator-BDNF-based neurotrophin therapy.

    Cell death and differentiation 2009;16;4;624-37

  • The EGFR is required for proper innervation to the skin.

    Maklad A, Nicolai JR, Bichsel KJ, Evenson JE, Lee TC, Threadgill DW and Hansen LA

    Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, Nebraska 68178, USA.

    EGFR family members are essential for proper peripheral nervous system development. A role for EGFR itself in peripheral nervous system development in vivo, however, has not been reported. We investigated whether EGFR is required for cutaneous innervation using Egfr null and skin-targeted Egfr mutant mice. Neuronal markers; including PGP9.5, GAP-43, acetylated tubulin, and neurofilaments; revealed that Egfr null dorsal skin was hyperinnervated with a disorganized pattern of innervation. In addition, receptor subtypes such as lanceolate endings were disorganized and immature. To determine whether the hyperinnervation phenotype resulted from a target-derived effect of loss of EGFR, mice lacking EGFR expression in the cutaneous epithelium were examined. These mice retained other aspects of the cutaneous Egfr null phenotype but exhibited normal innervation. The sensory deficits in Egfr null dorsal skin were not associated with any abnormality in the morphology or density of dorsal root ganglion (DRG) neurons or Schwann cells. However, explant and dissociated cell cultures of DRG revealed more extensive branching in Egfr null cultures. These data demonstrate that EGFR is required for proper cutaneous innervation during development and suggest that it limits axonal outgrowth and branching in a DRG-autonomous manner.

    Funded by: NCRR NIH HHS: 1 C06 RR17417-01, C06 RR017417, P20 RR-02-003, P20 RR018788, P20 RR018788-010001; NIEHS NIH HHS: R01 ES015585

    The Journal of investigative dermatology 2009;129;3;690-8

  • Initiation of olfactory placode development and neurogenesis is blocked in mice lacking both Six1 and Six4.

    Chen B, Kim EH and Xu PX

    Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine of NYU, New York, NY 10029, USA.

    Mouse olfactory epithelium (OE) originates from ectodermally derived placode, the olfactory placode that arises at the anterior end of the neural plate. Tissue grafting and recombination experiments suggest that the placode is derived from a common preplacodal domain around the neural plate and its development is directed by signals arising from the underlying mesoderm and adjacent neuroectoderm. In mice, loss of Six1 affects OE morphogenesis but not placode formation. We show here that embryos lacking both Six1 and Six4 failed to form the olfactory placode but the preplacodal region appeared to be specified as judged by the expression of Eya2, which marks the common preplacodal domain, suggesting a synergistic requirement of Six1 and Six4 in patterning the preplacodal ectoderm to a morphologic placode. Our results show that Six1 and Six4 are coexpressed in the preplacodal ectoderm from E8.0. In the olfactory pit, Six4 expression was observed in the peripheral precursors that overlap with Mash1-expressing cells, the early committed neuronal lineage. In contrast, Six1 is highly distributed in the peripheral regions where stem cells reside at E10.5 and it overlaps with Sox2 expression. Both genes are expressed in the basal and apical neuronal progenitors in the OE. Analyses of Six1;Six4 double mutant embryos demonstrated that the slightly thickened epithelium observed in the mutant was not induced for neuronal development. In contrast, in Six1(-/-) embryos, all neuronal lineage markers were initially expressed but the pattern of their expression was altered. Although very few, the pioneer neurons were initially present in the Six1 mutant OE. However, neurogenesis ceased by E12.5 due to markedly increased cell apoptosis and reduced proliferation, thus defining the cellular defects occurring in Six1(-/-) OE that have not been previously observed. Our findings demonstrate that Six1/4 function at the top of early events controlling olfactory placode formation and neuronal development. Our analyses show that the threshold of Six1/4 may be crucial for the expression of olfactory specific genes and that Six1 and Six4 may act synergistically to mediate olfactory placode specification and patterning through Fgf and Bmp signaling pathways.

    Funded by: NIDCD NIH HHS: R01 DC005824

    Developmental biology 2009;326;1;75-85

  • HDAC3 as a molecular chaperone for shuttling phosphorylated TR2 to PML: a novel deacetylase activity-independent function of HDAC3.

    Gupta P, Ho PC, Ha SG, Lin YW and Wei LN

    Institute of Microbial Technology, Chandigarh, India.

    TR2 is an orphan nuclear receptor specifically expressed in early embryos (Wei and Hsu, 1994), and a transcription factor for transcriptional regulation of important genes in stem cells including the gate keeper Oct4 (Park et al. 2007). TR2 is known to function as an activator (Wei et al. 2000), or a repressor (Chinpaisal et al., 1998, Gupta et al. 2007). Due to the lack of specific ligands, mechanisms triggering its activator or repressor function have remained puzzling for decades. Recently, we found that all-trans retinoic acid (atRA) triggers the activation of extracellular-signal-regulated kinase 2 (ERK2), which phosphorylates TR2 and stimulates its partitioning to promyelocytic leukemia (PML) nuclear bodies, thereby converting the activator function of TR2 into repression (Gupta et al. 2008; Park et al. 2007). Recruitment of TR2 to PML is a crucial step in the conversion of TR2 from an activator to a repressor. However, it is unclear how phosphorylated TR2 is recruited to PML, an essential step in converting TR2 from an activator to a repressor. In the present study, we use both in vitro and in vivo systems to address the problem of recruiting TR2 to PML nuclear bodies. First, we identify histone deacetylase 3 (HDAC3) as an effector molecule. HDAC3 is known to interact with TR2 (Franco et al. 2001) and this interaction is enhanced by the atRA-stimulated phosphorylation of TR2 at Thr-210 (Gupta et al. 2008). Secondly, in this study, we also find that the carrier function of HDAC3 is independent of its deacetylase activity. Thirdly, we find another novel activity of atRA that stimulates nuclear enrichment of HDAC3 to form nuclear complex with PML, which is ERK2 independent. This is the first report identifying a deacetylase-independent function for HDAC3, which serves as a specific carrier molecule that targets a specifically phosphorylated protein to PML NBs. This is also the first study delineating how protein recruitment to PML nuclear bodies occurs, which can be stimulated by atRA in an ERK2-independent manner. These findings could provide new insights into the development of potential therapeutics and in understanding how orphan nuclear receptor activities can be regulated without ligands.

    Funded by: NIDA NIH HHS: DA 11806, DA11190, K02 DA013926, K02-DA 13926, P50 DA011806, R01 DA011190; NIDDK NIH HHS: DK54733, DK60521, R01 DK054733, R01 DK060521

    PloS one 2009;4;2;e4363

  • Emx2 stimulates odorant receptor gene expression.

    McIntyre JC, Bose SC, Stromberg AJ and McClintock TS

    Department of Physiology, University of Kentucky, Lexington, KY 40536-0298, USA.

    The mechanisms selecting a single odorant receptor (OR) gene for expression in each olfactory sensory neuron (OSN) establish an OR expression pattern critical for odor discrimination. These mechanisms are largely unknown, but putative OR promoters contain homeodomain-like sites, implicating homeobox transcription factors such as Emx2. At embryonic day 18.5, expression of 49-76% of ORs was decreased in mice lacking Emx2, depending on the metric used. The decreases were due to fewer OSNs expressing each OR. Affected ORs showed changes that were disproportionately greater than the 42% reduction in mature neurons and similar decreases in unrelated olfactory neuron-enriched messenger RNAs in Emx2(-/-) mice. Both Class I and Class II ORs decreased, as did ORs expressed in both the dorsal and ventral regions of the epithelium. Conversely, 7% of Class II ORs tested were expressed more frequently, suggesting that some ORs are independent of Emx2. Emx2 helps stimulate transcription for many OR genes, which we hypothesize is through direct action at OR promoters, but Emx2 appears to have no significant role in regulating other aspects of OR gene expression, including the zonal patterns, OR gene cluster selection mechanisms, and singularity of OR gene choice.

    Funded by: NIDCD NIH HHS: R01 DC002736, R01 DC007194, R01DC002736, R01DC007194

    Chemical senses 2008;33;9;825-37

  • Rewiring the retinal ganglion cell gene regulatory network: Neurod1 promotes retinal ganglion cell fate in the absence of Math5.

    Mao CA, Wang SW, Pan P and Klein WH

    Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Medical School, Houston, TX 77030, USA.

    Retinal progenitor cells (RPCs) express basic helix-loop-helix (bHLH) factors in a strikingly mosaic spatiotemporal pattern, which is thought to contribute to the establishment of individual retinal cell identity. Here, we ask whether this tightly regulated pattern is essential for the orderly differentiation of the early retinal cell types and whether different bHLH genes have distinct functions that are adapted for each RPC. To address these issues, we replaced one bHLH gene with another. Math5 is a bHLH gene that is essential for establishing retinal ganglion cell (RGC) fate. We analyzed the retinas of mice in which Math5 was replaced with Neurod1 or Math3, bHLH genes that are expressed in another RPC and are required to establish amacrine cell fate. In the absence of Math5, Math5Neurod1-KI was able to specify RGCs, activate RGC genes and restore the optic nerve, although not as effectively as Math5. By contrast, Math5Math3-KI was much less effective than Math5Neurod1-KI in replacing Math5. In addition, expression of Neurod1 and Math3 from the Math5Neurod1-KI/Math3-KI allele did not result in enhanced amacrine cell production. These results were unexpected because they indicated that bHLH genes, which are currently thought to have evolved highly specialized functions, are nonetheless able to adjust their functions by interpreting the local positional information that is programmed into the RPC lineages. We conclude that, although Neurod1 and Math3 have evolved specialized functions for establishing amacrine cell fate, they are nevertheless capable of alternative functions when expressed in foreign environments.

    Funded by: NCI NIH HHS: CA016672; NEI NIH HHS: EY010608-139005, EY011930

    Development (Cambridge, England) 2008;135;20;3379-88

  • Sall1 regulates mitral cell development and olfactory nerve extension in the developing olfactory bulb.

    Harrison SJ, Nishinakamura R and Monaghan AP

    Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15261, USA.

    Sall1 is a zinc finger containing transcription factor that is highly expressed during mammalian embryogenesis. In humans, the developmental disorder Townes Brocks Syndrome is associated with mutations in the SALL1 gene. Sall1-deficient animals die at birth due to kidney deficits; however, its function in the nervous system has not been characterized. We examined the role of Sall1 in the developing olfactory system. We demonstrate that Sall1 is expressed by cells in the olfactory epithelium and olfactory bulb (OB). Sall1-deficient OBs are reduced in size and exhibit alterations in neurogenesis and mitral cell production. In addition, the olfactory nerve failed to extend past the ventral-medial region of the OB in Sall1-deficient animals. We observed intrinsic patterns of neurogenesis during olfactory development in control animals. In Sall1-mutant animals, these patterns of neurogenesis were disrupted. These findings suggest a role for Sall1 in regulating neuronal differentiation and maturation in developing neural structures.

    Funded by: NIAAA NIH HHS: R01AA13004; NIMH NIH HHS: 2R01MH60774, R01 MH060774, R01 MH060774-06, R01 MH060774-07, R01 MH060774-08

    Cerebral cortex (New York, N.Y. : 1991) 2008;18;7;1604-17

  • Endocannabinoid signaling controls pyramidal cell specification and long-range axon patterning.

    Mulder J, Aguado T, Keimpema E, Barabás K, Ballester Rosado CJ, Nguyen L, Monory K, Marsicano G, Di Marzo V, Hurd YL, Guillemot F, Mackie K, Lutz B, Guzmán M, Lu HC, Galve-Roperh I and Harkany T

    Department of Neuroscience, Retzius väg 8, and Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Scheeles väg 1, Karolinska Institutet, 17177 Stockholm, Sweden.

    Endocannabinoids (eCBs) have recently been identified as axon guidance cues shaping the connectivity of local GABAergic interneurons in the developing cerebrum. However, eCB functions during pyramidal cell specification and establishment of long-range axonal connections are unknown. Here, we show that eCB signaling is operational in subcortical proliferative zones from embryonic day 12 in the mouse telencephalon and controls the proliferation of pyramidal cell progenitors and radial migration of immature pyramidal cells. When layer patterning is accomplished, developing pyramidal cells rely on eCB signaling to initiate the elongation and fasciculation of their long-range axons. Accordingly, CB(1) cannabinoid receptor (CB(1)R) null and pyramidal cell-specific conditional mutant (CB(1)R(f/f,NEX-Cre)) mice develop deficits in neuronal progenitor proliferation and axon fasciculation. Likewise, axonal pathfinding becomes impaired after in utero pharmacological blockade of CB(1)Rs. Overall, eCBs are fundamental developmental cues controlling pyramidal cell development during corticogenesis.

    Funded by: Medical Research Council: MC_U117570528; NIDA NIH HHS: DA11322, DA15916, DA21696, K05 DA021696, P01 DA015916, R01 DA011322, R01 DA023214, R01DA023214; NIEHS NIH HHS: ES07332, T32 ES007332; NINDS NIH HHS: NS048884, R01 NS048884

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;25;8760-5

  • Identification of Phox2b-regulated genes by expression profiling of cranial motoneuron precursors.

    Pla P, Hirsch MR, Le Crom S, Reiprich S, Harley VR and Goridis C

    Ecole normale supérieure, Département de Biologie, 75005 Paris, France. Patrick.Pla@curie.u-psud.fr

    Background: Branchiomotor neurons comprise an important class of cranial motor neurons that innervate the branchial-arch-derived muscles of the face, jaw and neck. They arise in the ventralmost progenitor domain of the rhombencephalon characterized by expression of the homeodomain transcription factors Nkx2.2 and Phox2b. Phox2b in particular plays a key role in the specification of branchiomotor neurons. In its absence, generic neuronal differentiation is defective in the progenitor domain and no branchiomotor neurons are produced. Conversely, ectopic expression of Phox2b in spinal regions of the neural tube promotes cell cycle exit and neuronal differentiation and, at the same time, induces genes and an axonal phenotype characteristic for branchiomotor neurons. How Phox2b exerts its pleiotropic functions, both as a proneural gene and a neuronal subtype determinant, has remained unknown.

    Results: To gain further insights into the genetic program downstream of Phox2b, we searched for novel Phox2b-regulated genes by cDNA microarray analysis of facial branchiomotor neuron precursors from heterozygous and homozygous Phox2b mutant embryos. We selected for functional studies the genes encoding the axonal growth promoter Gap43, the Wnt antagonist Sfrp1 and the transcriptional regulator Sox13, which were not previously suspected to play roles downstream of Phox2b and whose expression was affected by Phox2b misexpression in the spinal cord. While Gap43 did not produce an obvious phenotype when overexpressed in the neural tube, Sfrp1 induced the interneuron marker Lhx1,5 and Sox13 inhibited neuronal differentiation. We then tested whether Sfrp1 and Sox13, which are down-regulated by Phox2b in the facial neuron precursors, would antagonize some aspects of Phox2b activity. Co-expression of Sfrp1 prevented Phox2b from repressing Lhx1,5 and alleviated the commissural axonal phenotype. When expressed together with Sox13, Phox2b was still able to promote cell cycle exit and neuronal differentiation, but the cells failed to relocate to the mantle layer and to extinguish the neural stem cell marker Sox2.

    Conclusion: Our results suggest novel roles for Sfrp1 and Sox13 in neuronal subtype specification and generic neuronal differentiation, respectively, and indicate that down-regulation of Sfrp1 and Sox13 are essential aspects of the genetic program controlled by Phox2b in cranial motoneurons.

    Neural development 2008;3;14

  • TNF-alpha in nucleus pulposus induces sensory nerve growth: a study of the mechanism of discogenic low back pain using TNF-alpha-deficient mice.

    Hayashi S, Taira A, Inoue G, Koshi T, Ito T, Yamashita M, Yamauchi K, Suzuki M, Takahashi K and Ohtori S

    Department of Orthopaedic Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan.

    We used retrograde neurotracing with fluoro-gold to investigate the relationship between tumor necrosis factor (TNF-á) and nerve growth into the nucleus pulposus (NP) of wild-type and TNF-alpha-deficient mice.

    Objective: To clarify mechanisms underlying nerve growth into the NP and the role of TNF-á in this process.

    Degeneration of lumbar intervertebral discs is a cause of low back pain. Pathogenesis may involve sensory nerve ingrowth into the inner layers and NP of degenerating discs. We hypothesized that TNF-á in the NP is a major inducer of nerve ingrowth and investigated this hypothesis in vivo using wild-type and TNF-á-deficient mice.

    Methods: NP was harvested at the L4/5 level from 10 wild-type and 10 TNF-deficient mice. These 20 samples of wild-type NP or TNF-deficient NP were mixed with fluoro-gold and injected into the left quadriceps muscle of 20 other wild-type mice (1 sample per mouse). Five control mice underwent sham operations in which they received similar injections of NP-free fluoro-gold into their left quadriceps muscles to detect whether neurons innervating the muscle establish contact with injected NP. Seven and 14 days after surgery, left L4 dorsal root ganglions were removed and incubated with antibodies against growth-associated protein 43 (GAP43), a marker of axonal growth. We evaluated fluoro-gold-labeled and GAP43-immunoreactive dorsal root ganglions neurons.

    Results: Within the set of fluoro-gold-labeled neurons, 10% were positive for GAP43 in sham-operated animals, 22% positive in the TNF-deficient NP group, and 38% positive in the wild-type NP group. These intergroup differences in the percentage of GAP43-positive neurons were statistically significant (sham vs. TNF-deficient NP group: P = 0.009; TNF-deficient NP group vs wild-type NP group: P = 0.026). CONCLUSION.: The percentage of fluoro-gold-labeled GAP43-immunoreactive neurons significantly increased after injections of NP harvested from both mouse types. Furthermore, the percentage of GAP43-immunoreactive neurons was significantly higher in mice receiving wild-type NP compared with mice receiving TNF-deficient NP. These findings suggest that TNF-alpha acts as an inducer of axonal growth into degenerated discs, as evidenced by decreased GAP-43 immunoreactivity in mice receiving TNF-deficient NP injections and even lower GAP-43 immunoreactivity in control mice receiving NP-free fluoro-gold injections.

    Spine 2008;33;14;1542-6

  • ISL1 and BRN3B co-regulate the differentiation of murine retinal ganglion cells.

    Pan L, Deng M, Xie X and Gan L

    Department of Ophthalmology, University of Rochester, Rochester, NY 14642, USA.

    LIM-homeodomain (HD) and POU-HD transcription factors play crucial roles in neurogenesis. However, it remains largely unknown how they cooperate in this process and what downstream target genes they regulate. Here, we show that ISL1, a LIM-HD protein, is co-expressed with BRN3B, a POU-HD factor, in nascent post-mitotic retinal ganglion cells (RGCs). Similar to the Brn3b-null retinas, retina-specific deletion of Isl1 results in the apoptosis of a majority of RGCs and in RGC axon guidance defects. The Isl1 and Brn3b double null mice display more severe retinal abnormalities with a near complete loss of RGCs, indicating the synergistic functions of these two factors. Furthermore, we show that both Isl1 and Brn3b function downstream of Math5 to regulate the expression of a common set of RGC-specific genes. Whole-retina chromatin immunoprecipitation and in vitro transactivation assays reveal that ISL1 and BRN3B concurrently bind to and synergistically regulate the expression of a common set of RGC-specific genes. Thus, our results uncover a novel regulatory mechanism of BRN3B and ISL1 in RGC differentiation.

    Funded by: NEI NIH HHS: EY013426, EY015551, R01 EY013426, R01 EY013426-06A1, R01 EY013426-07, R01 EY015551, R01 EY015551-04

    Development (Cambridge, England) 2008;135;11;1981-90

  • Synaptogenesis in the mouse olfactory bulb during glomerulus development.

    Blanchart A, Romaguera M, García-Verdugo JM, de Carlos JA and López-Mascaraque L

    Department of Cellular, Molecular and Developmental Neurobiology, Instituto Cajal, CSIC, Madrid, Spain.

    Synaptogenesis is essential for the development of neuronal networks in the brain. In the olfactory bulb (OB) glomeruli, numerous synapses must form between sensory olfactory neurons and the dendrites of mitral/tufted and periglomerular cells. Glomeruli develop from E13 to E16 in the mouse, coincident with an increment of the neuropil in the border between the external plexiform (EPL) and olfactory nerve layers (ONL), coupled to an extensive labelling of phalloidin and GAP-43 from the ONL to EPL. We have tracked synaptogenesis in the OB during this period by electron microscopy (EM) and immunolabelling of the transmembrane synaptic vesicle glycoprotein SV-2. No SV-2 labelling or synapses were detected at E13, although electrodense junctions lacking synaptic vesicles could be observed by EM. At E14, sparse SV-2 labelling appears in the most ventral and medial part of the incipient OB, which displays a ventro-dorsal gradient by E15 but covers the entire OB by E16. These data establish a spatio-temporal pattern of synaptogenesis, which perfectly matches with the glomeruli formation in developing OB.

    The European journal of neuroscience 2008;27;11;2838-46

  • Foxg1 is required for development of the vertebrate olfactory system.

    Duggan CD, DeMaria S, Baudhuin A, Stafford D and Ngai J

    Department of Molecular and Cell Biology, Helen Wills Neuroscience Institute, and Functional Genomics Laboratory, University of California, Berkeley, California 94720-3200, USA.

    Illuminating the molecular identity and regulation of early progenitor cells in the olfactory sensory epithelium represents an important challenge in the field of neural development. We show in both mouse and zebrafish that the winged helix transcription factor Foxg1 is expressed in an early progenitor population of the olfactory placode. In the mouse, Foxg1 is first expressed throughout the olfactory placode but later becomes restricted to the ventrolateral olfactory epithelium. The essential role of Foxg1 in olfactory development is demonstrated by the strikingly severe phenotype of Foxg1 knock-out mice: older embryos have no recognizable olfactory structures, including epithelium, bulb, or vomeronasal organs. Initially, a small number of olfactory progenitors are specified but show defects in both proliferation and differentiation. Similarly, antisense RNA knockdown of Foxg1 expression in the zebrafish shows a reduction in the number of neurons and mitotic cells in olfactory rosettes, mirroring the phenotype seen in the mouse Foxg1 null mutant. Using mosaic analysis in the zebrafish, we show that Foxg1 is required cell-autonomously for the production of mature olfactory receptor neurons. Therefore, we identified an evolutionarily conserved requirement for Foxg1 in the development of the vertebrate olfactory system.

    Funded by: NIDCD NIH HHS: R01 DC007235, R01 DC007235-01, R01 DC007235-02, R01 DC007235-03, R01 DC007235-04

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;20;5229-39

  • Gene regulation logic in retinal ganglion cell development: Isl1 defines a critical branch distinct from but overlapping with Pou4f2.

    Mu X, Fu X, Beremand PD, Thomas TL and Klein WH

    Department of Biochemistry and Molecular Biology, M. D. Anderson Cancer Center and Graduate Training Program in Genes and Development, Graduate School of Biomedical Sciences, University of Texas, Houston, TX 77030, USA. xiumu@mdanderson.org

    Understanding gene regulatory networks (GRNs) that control neuronal differentiation will provide systems-level perspectives on neurogenesis. We have previously constructed a model for a GRN in retinal ganglion cell (RGC) differentiation in which four hierarchical tiers of transcription factors ultimately control the expression of downstream terminal genes. Math5 occupies a central node in the hierarchy because it is essential for the formation of RGCs and the expression of the immediate downstream factor Pou4f2. Based on its expression, we also proposed that Isl1, a LIM-homeodomain factor, functions in parallel with Pou4f2 and downstream of Math5 in the RGC GRN. To determine whether this was the case, a conditional Isl1 allele was generated and deleted specifically in the developing retina. Although RGCs formed in Isl1-deleted retinas, most underwent apoptosis, and few remained at later stages. By microarray analysis, we identified a distinct set of genes whose expression depended on Isl1. These genes are all downstream of Math5, and some of them, but not all, also depend on Pou4f2. Additionally, Isl1 was required for the sustained expression of Pou4f2, suggesting that Isl1 positively regulates Pou4f2 after Math5 levels are diminished. The results demonstrate an essential role for Isl1 in RGC development and reveal two distinct but intersecting branches of the RGC GRN downstream of Math5, one directed by Pou4f2 and the other by Isl1. They also reveal that identical RGC expression patterns are achieved by different combinations of divergent inputs from upstream transcription factors.

    Funded by: NCI NIH HHS: CA16672, P30 CA016672; NEI NIH HHS: EY010608, EY011930, P30 EY010608, R01 EY011930

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;19;6942-7

  • Robos and slits control the pathfinding and targeting of mouse olfactory sensory axons.

    Nguyen-Ba-Charvet KT, Di Meglio T, Fouquet C and Chédotal A

    Université Pierre et Marie Curie and Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7102, F-75005 Paris, France.

    Odorants are detected by olfactory receptor neurons (ORNs) located in the olfactory epithelium. In mice, ORNs expressing the same odorant receptor (OR) project to a single glomerulus out of 1800 in the olfactory bulb (OB). It has been proposed that OR-derived cAMP signals guide ORN axons to their glomeruli rather than OR themselves. Recently, it has also been shown that the axon guidance molecule Slit1 and its receptor Robo2 control the dorsoventral segregation of ORN axons as they are projecting to the OB. We have analyzed the development of olfactory projections in Slit1/Slit2 and Robo1/Robo2 single and double mutants. We show that in Robo1-/-;Robo2-/- mice, most ORN axons fail to enter the OB and instead project caudally into the diencephalon. Moreover, in these mice, ORN axons expressing the same OR project to several glomeruli at ectopic positions. Thus, Slit1, Slit2, Robo1, and Robo2 cooperate to control the convergence of ORN axons to the OB and the precise targeting of ORN axons to specific glomeruli.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;16;4244-9

  • Axonal regeneration after optic nerve crush in Nogo-A/B/C knockout mice.

    Su Y, Wang F, Zhao SG, Pan SH, Liu P, Teng Y and Cui H

    Department of Ophthalmology, First Clinical College of Harbin Medical University, Harbin, China.

    Purpose: The axonal regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) crush was investigated both in vivo and in vitro on Nogo-A/B/C knockout mice.

    Methods: The study used 20 Nogo-A/B/C knockout mice in the experimental group, and 20 C57BL/6 mice in the control group. Partial ON injury was induced by using a specially designed ON clip to pinch the ON 1 mm behind the mouse eyeball with 40 g pressure for 9 s. The left ON was injured in both groups, but the right ON was left untouched in the control group. Nogo-A/B/C mRNA was studied by in situ hybridization in both groups. GAP-43 was studied by immunofluorescence staining on frozen sections. RGCs were purified and cultured in DMEM medium containing B-27. Cells were then immunostained with both Thy1.1 and GAP-43 antibodies. The axonal growth of RGCs was calculated by a computerized image analyzer.

    Results: GAP-43 expression was significantly higher in the experimental group than in the control group (p<0.01). GAP-43 antibody binding was demonstrated in the axons of cultured RGCs. Axonal growth was significantly more active at every observed time point in the experimental group than in the control group (F=43.25, 32.16; p<0.01).

    Conclusions: Nogo genes play an inhibitive role in the axonal regeneration after ON injury, while Nogo-knockout is an effective way to eliminate this inhibition and accelerate axonal regeneration.

    Molecular vision 2008;14;268-73

  • GAP-43 is key to mitotic spindle control and centrosome-based polarization in neurons.

    Mishra R, Gupta SK, Meiri KF, Fong M, Thostrup P, Juncker D and Mani S

    National Brain Research Center, Manesar, Haryana, India.

    In neurons, the position of the centrosome during final mitosis marks the point of emergence of the future axon. However, the molecular underpinnings linking centrosome position to axon emergence are unknown. GAP-43 is a calmodulin-binding IQ motif protein that regulates neuronal cytoskeletal architecture by interacting with F-actin in a phosphorylation dependent manner. Here we show that GAP-43 is associated with the centrosome and plays a critical role in mitosis and acquisition of neuronal polarity in cerebellar granule neurons. In the absence of GAP-43, the centrosome position is delinked from process outgrowth and is only capable of mediating morphological polarization, however molecular specification of the axonal compartment does not take place. These results show that GAP-43 is required to link centrosome position to process outgrowth in order to generate neuronal polarity in cerebellar granule cells.

    Funded by: NINDS NIH HHS: R01NS33118; PHS HHS: R03TWO6050

    Cell cycle (Georgetown, Tex.) 2008;7;3;348-57

  • Six3 inactivation causes progressive caudalization and aberrant patterning of the mammalian diencephalon.

    Lavado A, Lagutin OV and Oliver G

    Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794, USA.

    The homeobox gene Six3 represses Wnt1 transcription. It is also required in the anterior neural plate for the development of the mammalian rostral forebrain. We have now determined that at the 15- to 17-somite stage, the prospective diencephalon is the most-anterior structure in the Six3-null brain, and Wnt1 expression is anteriorly expanded. Consequently, the brain caudalizes, and at the 22- to 24-somite stage, the prospective thalamic territory is the most-anterior structure. At around E11.0, the pretectum replaces this structure. Analysis of Six3;Wnt1 double-null mice revealed that Six3-mediated repression of Wnt1 is necessary for the formation of the rostral diencephalon and that Six3 activity is required for the formation of the telencephalon. These results provide insight into the mechanisms that establish anteroposterior identity in the developing mammalian brain.

    Funded by: NCI NIH HHS: CA-21765; NEI NIH HHS: EY12162

    Development (Cambridge, England) 2008;135;3;441-50

  • N-myc coordinates retinal growth with eye size during mouse development.

    Martins RA, Zindy F, Donovan S, Zhang J, Pounds S, Wey A, Knoepfler PS, Eisenman RN, Roussel MF and Dyer MA

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

    Myc family members play crucial roles in regulating cell proliferation, size, differentiation, and survival during development. We found that N-myc is expressed in retinal progenitor cells, where it regulates proliferation in a cell-autonomous manner. In addition, N-myc coordinates the growth of the retina and eye. Specifically, the retinas of N-myc-deficient mice are hypocellular but are precisely proportioned to the size of the eye. N-myc represses the expression of the cyclin-dependent kinase inhibitor p27Kip1 but acts independently of cyclin D1, the major D-type cyclin in the developing mouse retina. Acute inactivation of N-myc leads to increased expression of p27Kip1, and simultaneous inactivation of p27Kip1 and N-myc rescues the hypocellular phenotype in N-myc-deficient retinas. N-myc is not required for retinal cell fate specification, differentiation, or survival. These data represent the first example of a role for a Myc family member in retinal development and the first characterization of a mouse model in which the hypocellular retina is properly proportioned to the other ocular structures. We propose that N-myc lies upstream of the cell cycle machinery in the developing mouse retina and thus coordinates the growth of both the retina and eye through extrinsic cues.

    Genes & development 2008;22;2;179-93

  • The protein kinase C phosphorylation site on GAP-43 differentially regulates information storage.

    Holahan M and Routtenberg A

    Department of Psychology and Institute of Neuroscience, Carleton University, Ottawa, Canada. matthew_holahan@carleton.ca

    Protein kinase C (PKC) is known to regulate phosphorylation of substrates such as MARCKS, GAP-43, and the NMDA receptor, all of which have been linked to synaptic plasticity underlying information storage processes. Here we report on three transgenic mice isoforms differentiated both by mutation of the PKC site on GAP-43 as well as by their performance in three learning situations: (1) a radial arm maze task, which evaluates spatial memory and its retention, (2) fear conditioning which assesses contextual memory, and (3) the water maze which also evaluates spatial memory and its retention. The present results show, for the first time to our knowledge, that the phosphorylation state of a single site on an identified brain growth- and plasticity-associated protein differentially regulates performance of three different memory-associated tasks.

    Funded by: NIA NIH HHS: AG20506; NIMH NIH HHS: MH65436, R01 MH065436, R01 MH065436-05

    Hippocampus 2008;18;11;1099-102

  • Alterations in mossy fiber physiology and GAP-43 expression and function in transgenic mice overexpressing HuD.

    Tanner DC, Qiu S, Bolognani F, Partridge LD, Weeber EJ and Perrone-Bizzozero NI

    Department of Neurosciences, University of New Mexico HSC, Albuquerque, New Mexico 87106, USA.

    HuD is a neuronal RNA-binding protein associated with the stabilization of mRNAs for GAP-43 and other neuronal proteins that are important for nervous system development and learning and memory mechanisms. To better understand the function of this protein, we generated transgenic mice expressing human HuD (HuD-Tg) in adult forebrain neurons. We have previously shown that expression of HuD in adult dentate granule cells results in an abnormal accumulation of GAP-43 mRNA via posttranscriptional mechanisms. Here we show that this mRNA accumulation leads to the ectopic expression of GAP-43 protein in mossy fibers. Electrophysiological analyses of the mossy fiber to CA3 synapse of HuD-Tg mice revealed increases in paired-pulse facilitation (PPF) at short interpulse intervals and no change in long-term potentiation (LTP). Presynaptic calcium transients at the same synapses exhibited faster time constants of decay, suggesting a decrease in the endogenous Ca(2+) buffer capacity of mossy fiber terminals of HuD-Tg mice. Under resting conditions, GAP-43 binds very tightly to calmodulin sequestering it and then releasing it upon PKC-dependent phosphorylation. Therefore, subsequent studies examined the extent of GAP-43 phosphorylation and its association to calmodulin. We found that despite the increased GAP-43 expression in HuD-Tg mice, the levels of PKC-phosphorylated GAP-43 were decreased in these animals. Furthermore, in agreement with the increased proportion of nonphosphorylated GAP-43, HuD-Tg mice showed increased binding of calmodulin to this protein. These results suggest that a significant amount of calmodulin may be trapped in an inactive state, unable to bind free calcium, and activate downstream signaling pathways. In conclusion, we propose that an unregulated expression of HuD disrupts mossy fiber physiology in adult mice in part by altering the expression and phosphorylation of GAP-43 and the amount of free calmodulin available at the synaptic terminal.

    Funded by: NIA NIH HHS: AG022574, R01 AG022574, R01 AG022574-05; NIAAA NIH HHS: T32 AA1427; NINDS NIH HHS: NS30255, R01 NS030255, R01 NS030255-16

    Hippocampus 2008;18;8;814-23

  • Both cell-autonomous and cell non-autonomous functions of GAP-43 are required for normal patterning of the cerebellum in vivo.

    Shen Y, Mishra R, Mani S and Meiri KF

    Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.

    Growth-associated protein 43 (GAP-43) is required for development of a functional cerebral cortex in vertebrates; however, its role in cerebellar development is not well understood. Recently, we showed that absence of GAP-43 caused defects in proliferation, differentiation, and polarization of cerebellar granule cells. In this paper, we show that absence of GAP-43 causes defects in cerebellar patterning that reflect both cell-autonomous and non-autonomous functions. Cell-autonomous effects of GAP-43 impact precursor proliferation and axon targeting: In its absence, (1) proliferation of granule cell precursors in response to sonic hedgehog and fibroblast growth factor is inhibited, (2) proliferation of neuroepithelial precursors is inhibited, and (3) targeting of climbing fibers to the central lobe is disrupted. Cell non-autonomous effects of GAP-43 impact differentiated Purkinje cells in which GAP-43 has been downregulated: In its absence, both maturation and mediolateral patterning of Purkinje cells are inhibited. Both cell-autonomous and non-autonomous functions of GAP-43 involve its phosphorylation by protein kinase C. GAP-43 is phosphorylated in granule cell precursors in response to sonic hedgehog in vitro, and phosphorylated GAP-43 is also found in proliferating neuroepithelium and climbing fibers. Phosphorylated GAP-43 is specifically enriched in the presynaptic terminals of parallel and climbing fibers that innervate Purkinje cell bodies and dendrites. The cell-autonomous and non-autonomous effects of GAP-43 converge on the central lobe. The multiple effects of GAP-43 on cerebellar development suggest that it is a critical downstream transducer of signaling mechanisms that integrate generation of cerebellar structure with functional parcellation at the central lobe.

    Funded by: FIC NIH HHS: R03 TW006050, R03TW0605; NINDS NIH HHS: NS33118, R01 NS033118

    Cerebellum (London, England) 2008;7;3;451-66

  • Characterization of connexin30.3-deficient mice suggests a possible role of connexin30.3 in olfaction.

    Zheng-Fischhöfer Q, Schnichels M, Dere E, Strotmann J, Loscher N, McCulloch F, Kretz M, Degen J, Reucher H, Nagy JI, Peti-Peterdi J, Huston JP, Breer H and Willecke K

    Institut für Genetik, Abteilung Molekulargenetik, Römerstrassae 164, D-53117 Bonn, Germany.

    We have generated connexin30.3-deficient mice in which the coding region of the connexin30.3 gene was replaced by the lacZ reporter gene. The expression pattern of this connexin was characterized using beta-galactosidase staining and immunoblot analyses. In skin, beta-galactosidase/connexin30.3 protein was expressed in the spinous and granulous layers of the epidermis. Specific beta-galactosidase/connexin30.3 expression was also detected in the thin ascending limb of Henle's loop in the kidney. In addition, we found beta-galactosidase/connexin30.3 in progenitor cells of the olfactory epithelium and in a subpopulation of cells in the apical layer of the vomeronasal organ. Connexin30.3-deficient mice were fertile and displayed no abnormalities in the skin or in the chemosensory systems. Furthermore, they showed normal auditory thresholds as measured by brain stem evoked potentials. These mice did, however, exhibit reduced behavioural responses to a vanilla scent.

    Funded by: NIDDK NIH HHS: R01 DK064324, R01 DK064324-04

    European journal of cell biology 2007;86;11-12;683-700

  • Six1 is essential for early neurogenesis in the development of olfactory epithelium.

    Ikeda K, Ookawara S, Sato S, Ando Z, Kageyama R and Kawakami K

    Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan.

    The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, "early neurogenesis" occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows "established neurogenesis," in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1-/-). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1-/- at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1-/-. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5.

    Developmental biology 2007;311;1;53-68

  • Neurofibromatosis-1 regulates neuronal and glial cell differentiation from neuroglial progenitors in vivo by both cAMP- and Ras-dependent mechanisms.

    Hegedus B, Dasgupta B, Shin JE, Emnett RJ, Hart-Mahon EK, Elghazi L, Bernal-Mizrachi E and Gutmann DH

    Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA.

    Individuals with neurofibromatosis type 1 (NF1) develop abnormalities of both neuronal and glial cell lineages, suggesting that the NF1 protein neurofibromin is an essential regulator of neuroglial progenitor function. In this regard, Nf1-deficient embryonic telencephalic neurospheres exhibit increased self-renewal and prolonged survival as explants in vivo. Using a newly developed brain lipid binding protein (BLBP)-Cre mouse strain to study the role of neurofibromin in neural progenitor cell function in the intact animal, we now show that neuroglial progenitor Nf1 inactivation results in increased glial lineage proliferation and abnormal neuronal differentiation in vivo. Whereas the glial cell lineage abnormalities are recapitulated by activated Ras or Akt expression in vivo, the neuronal abnormalities were Ras- and Akt independent and reflected impaired cAMP generation in Nf1-deficient cells in vivo and in vitro. Together, these findings demonstrate that neurofibromin is required for normal glial and neuronal development involving separable Ras-dependent and cAMP-dependent mechanisms.

    Funded by: NCI NIH HHS: 1-UO1-CA84314; NIDDK NIH HHS: R03 DK068028, R03 DK068028-01

    Cell stem cell 2007;1;4;443-57

  • Diffusion tensor magnetic resonance imaging and tract-tracing analysis of Probst bundle structure in Netrin1- and DCC-deficient mice.

    Ren T, Zhang J, Plachez C, Mori S and Richards LJ

    Department of Anatomy and Neurobiology and The Program in Neuroscience, The University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

    In many cases of callosal dysgenesis in both human patients and mouse models, misguided fibers from the cortex form abnormal bilateral, barrel-shaped structures known as Probst bundles. Because little is known about how axons are arranged within these anomalous fiber bundles, understanding this arrangement may provide structural and molecular insights into how axons behave when they are misguided in vivo. Previous studies described these bundles as longitudinal swirls of axons that fail to cross the midline (Ozaki et al., 1987). However, recent studies on human acallosal patients using diffusion tensor magnetic resonance imaging (DTMRI) technology suggest that axons project in an anteroposterior direction within the Probst bundle (Lee et al., 2004; Tovar-Moll et al., 2007). This led us to ask the question, is DTMRI an accurate method for analyzing axonal tracts in regions of high axon overlap and disorganization, or is our current perception of axon arrangement within these bundles inaccurate? Using DTMRI, immunohistochemistry, and carbocyanine dye tract-tracing studies, we analyzed the Probst bundles in both Netrin1 and deleted in colorectal cancer (DCC) mutant mice. Our findings indicate that DTMRI can accurately demonstrate fiber tract orientation and morphology where axons are in ordered arrays such as in the dorsal part of the bundle. In ventral areas, where the axons are disorganized, no coordinated diffusion is apparent via DTMRI. In these regions, a higher-resolution approach such as tract tracing is required. We conclude that in DCC and Netrin1 mutant mice, guidance mechanisms remain in the dorsal part of the tract but are lost ventrally.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;39;10345-9

  • ETS transcription factor Erm controls subsynaptic gene expression in skeletal muscles.

    Hippenmeyer S, Huber RM, Ladle DR, Murphy K and Arber S

    Biozentrum, Department of Cell Biology, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.

    Accumulation of specific proteins at synaptic structures is essential for synapse assembly and function, but mechanisms regulating local protein enrichment remain poorly understood. At the neuromuscular junction (NMJ), subsynaptic nuclei underlie motor axon terminals within extrafusal muscle fibers and are transcriptionally distinct from neighboring nuclei. In this study, we show that expression of the ETS transcription factor Erm is highly concentrated at subsynaptic nuclei, and its mutation in mice leads to severe downregulation of many genes with normally enriched subsynaptic expression. Erm mutant mice display an expansion of the muscle central domain in which acetylcholine receptor (AChR) clusters accumulate, show gradual fragmentation of AChR clusters, and exhibit symptoms of muscle weakness mimicking congenital myasthenic syndrome (CMS). Together, our findings define Erm as an upstream regulator of a transcriptional program selective to subsynaptic nuclei at the NMJ and underscore the importance of transcriptional control of local synaptic protein accumulation.

    Neuron 2007;55;5;726-40

  • EUCOMM--the European conditional mouse mutagenesis program.

    Friedel RH, Seisenberger C, Kaloff C and Wurst W

    GSF-National Research Center for Environment and Health, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.

    Functional analysis of the mammalian genome is an enormous challenge for biomedical scientists. To facilitate this endeavour, the European Conditional Mouse Mutagenesis Program (EUCOMM) aims at generating up to 12 000 mutations by gene trapping and up to 8000 mutations by gene targeting in mouse embryonic stem (ES) cells. These mutations can be rendered into conditional alleles, allowing Cre recombinase-mediated disruption of gene function in a time- and tissue-specific manner. Furthermore, the EUCOMM program will generate up to 320 mouse lines from the EUCOMM resource and up to 20 new Cre driver mouse lines. The EUCOMM resource of vectors, mutant ES cell lines and mutant mice will be openly available to the scientific community. EUCOMM will be one of the cornerstones of an international effort to create a global mouse mutant resource.

    Briefings in functional genomics & proteomics 2007;6;3;180-5

  • Increased thalamocortical synaptic response and decreased layer IV innervation in GAP-43 knockout mice.

    Albright MJ, Weston MC, Inan M, Rosenmund C and Crair MC

    Department of Neuroscience, Baylor College of Medicine, Houston, Texas, USA.

    The growth-associated protein, GAP-43, is an axonally localized neuronal protein with high expression in the developing brain and in regenerating neurites. Mice that lack GAP-43 (GAP-43 -/-) fail to form a whisker-related barrel map. In this study, we use GAP-43 -/- mice to examine GAP-43 synaptic function in the context of thalamocortical synapse development and cortical barrel map formation. Examination of thalamocortical synaptic currents in an acute brain slice preparation and in autaptic thalamic neurons reveals that GAP-43 -/- synapses have larger alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptor (AMPAR)-mediated currents than controls despite similar AMPAR function and normal probability of vesicular release. Interestingly, GAP-43 -/- synapses are less sensitive to blockade by a competitive glutamate receptor antagonist, suggesting higher levels of neurotransmitter in the cleft during synaptic transmission. Field excitatory postsynaptic potentials (EPSPs) from GAP-43 -/- thalamocortical synapses reveal a reduced fiber response, and anatomical analysis shows reduced thalamic innervation of barrel cortex in GAP-43 -/- mice. Despite this fact synaptic responses in the field EPSPs are similar in GAP-43 -/- mice and wild-type littermate controls, and the ratio of AMPAR-mediated to N-methyl-d-aspartate receptor (NMDAR)-mediated currents (AMPAR:NMDAR ratio) is larger than normal. This suggests that GAP-43 -/- mice form fewer thalamocortical synapses in layer IV because of decreased anatomical innervation of the cortex, but the remaining contacts are individually stronger possibly due to increased neurotransmitter concentration in the synaptic cleft. Together, these results indicate that in addition to its well known role in axonal pathfinding GAP-43 plays a functional role in regulating neurotransmitter release.

    Funded by: NEI NIH HHS: EY-15788; NIMH NIH HHS: MH-62639, R01 MH062639; NINDS NIH HHS: NS-050655

    Journal of neurophysiology 2007;98;3;1610-25

  • Dlx transcription factors promote migration through repression of axon and dendrite growth.

    Cobos I, Borello U and Rubenstein JL

    Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94158, USA. inma.cobos@ucsf.edu

    In the mouse telencephalon, Dlx homeobox transcription factors are essential for the tangential migration of subpallial-derived GABAergic interneurons to neocortex. However, the mechanisms underlying this process are poorly understood. Here, we demonstrate that Dlx1/2 has a central role in restraining neurite growth of subpallial-derived immature interneurons at a stage when they migrate tangentially to cortex. In Dlx1-/-;Dlx2-/- mutants, neurite length is increased and cells fail to migrate. In Dlx1-/-;Dlx2+/- mutants, while the tangential migration of immature interneurons appears normal, they develop dendritic and axonal processes with increased length and decreased branching, and have deficits in their neocortical laminar positions. Thus, Dlx1/2 is required for coordinating programs of neurite maturation and migration. In this regard, we provide genetic evidence that in immature interneurons Dlx1/2 repression of the p21-activated serine/threonine kinase PAK3, a downstream effector of the Rho family of GTPases, is critical in restraining neurite growth and promoting tangential migration.

    Funded by: NIMH NIH HHS: K05 MH065670, R01 MH049428, R01 MH49428

    Neuron 2007;54;6;873-88

  • Molecular heterogeneity of developing retinal ganglion and amacrine cells revealed through single cell gene expression profiling.

    Trimarchi JM, Stadler MB, Roska B, Billings N, Sun B, Bartch B and Cepko CL

    Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

    During development of the central nervous system (CNS), cycling uncommitted progenitor cells give rise to a variety of distinct neuronal and glial cell types. As these different cell types are born they progress from newly specified cells to fully differentiated neurons and glia. In order to define the developmental processes of individual cell types, single cell expression profiling was carried out on developing ganglion and amacrine cells of the murine retina. Individual cells from multiple developmental stages were isolated and profiled on Affymetrix oligonucleotide arrays. Two-color fluorescent in situ hybridization on dissociated retinas was used to verify and extend the microarray results by allowing quantitative measurements of a large number of cells coexpressing two genes. Together, these experiments have yielded an expanded view of the processes underway in developing retinal ganglion and amacrine cells, as well as several hundred new marker genes for these cell types. In addition, this study has allowed for the definition of some of the molecular heterogeneity both between developing ganglion and amacrine cells and among subclasses of each cell type.

    Funded by: NEI NIH HHS: EY08064, F32 EY014495, T32 EY007145

    The Journal of comparative neurology 2007;502;6;1047-65

  • Plexin-B2, but not Plexin-B1, critically modulates neuronal migration and patterning of the developing nervous system in vivo.

    Deng S, Hirschberg A, Worzfeld T, Penachioni JY, Korostylev A, Swiercz JM, Vodrazka P, Mauti O, Stoeckli ET, Tamagnone L, Offermanns S and Kuner R

    Institute of Pharmacology, University of Heidelberg, 69120 Heidelberg, Germany.

    Semaphorins and their receptors, plexins, have emerged as important cellular cues regulating key developmental processes. B-type plexins directly regulate the actin cytoskeleton in a variety of cell types. Recently, B-type plexins have been shown to be expressed in striking patterns in the nervous system over critical developmental windows. However, in contrast to the well characterized plexin-A family, the functional role of plexin-B proteins in neural development and organogenesis in vertebrates in vivo is not known. Here, we have elucidated the functional contribution of the two neuronally expressed plexin-B proteins, Plexin-B1 or Plexin-B2, toward the development of the peripheral nervous system and the CNS by generating and analyzing constitutive knock-out mice. The development of the nervous system was found to be normal in mice lacking Plexin-B1, whereas mice lacking Plexin-B2 demonstrated defects in closure of the neural tube and a conspicuous disorganization of the embryonic brain. After analyzing mutant mice, which bypassed neural tube defects, we observed a key requirement for Plexin-B2 in proliferation and migration of granule cell precursors in the developing dentate gyrus, olfactory bulb, and cerebellum. Furthermore, we identified semaphorin 4C as a high-affinity ligand for Plexin-B2 in binding and functional assays. Semaphorin 4C stimulated activation of ErbB-2 and RhoA via Plexin-B2 and enhanced proliferation and migration of granule cell precursors. Semaphorin 4C-induced proliferation of ventricular zone neuroblasts was abrogated in mice lacking Plexin-B2. These genetic and functional analyses reveal a key requirement for Plexin-B2, but not Plexin-B1, in patterning of the vertebrate nervous system in vivo.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;23;6333-47

  • GAP-43 gene expression regulates information storage.

    Holahan MR, Honegger KS, Tabatadze N and Routtenberg A

    Department of Psychology and Neurobiology, Northwestern University Interdepartmental Neuroscience Program, Northwestern University, Evanston, Illinois 60208, USA. matthew_holahan@carleton.ca

    Previous reports have shown that overexpression of the growth- and plasticity-associated protein GAP-43 improves memory. However, the relation between the levels of this protein to memory enhancement remains unknown. Here, we studied this issue in transgenic mice (G-Phos) overexpressing native, chick GAP-43. These G-Phos mice could be divided at the behavioral level into "spatial bright" and "spatial dull" groups based on their performance on two hidden platform water maze tasks. G-Phos dull mice showed both acquisition and retention deficits on the fixed hidden platform task, but were able to learn a visible platform task. G-Phos bright mice showed memory enhancement relative to wild type on the more difficult movable hidden platform spatial memory task. In the hippocampus, the G-Phos dull group showed a 50% greater transgenic GAP-43 protein level and a twofold elevated transgenic GAP-43 mRNA level than that measured in the G-Phos bright group. Unexpectedly, the dull group also showed an 80% reduction in hippocampal Tau1 staining. The high levels of GAP-43 seen here leading to memory impairment find its histochemical and behavioral parallel in the observation of Rekart et al. (Neuroscience 126: 579-584) who described elevated levels of GAP-43 protein in the hippocampus of Alzheimer's patients. The present data suggest that moderate overexpression of a phosphorylatable plasticity-related protein can enhance memory, while excessive overexpression may produce a "neuroplasticity burden" leading to degenerative and hypertrophic events culminating in memory dysfunction.

    Funded by: NIA NIH HHS: AG20506, T32 AG020506; NIMH NIH HHS: MH65436, R01 MH065436

    Learning & memory (Cold Spring Harbor, N.Y.) 2007;14;6;407-15

  • Hand2 is necessary for terminal differentiation of enteric neurons from crest-derived precursors but not for their migration into the gut or for formation of glia.

    D'Autréaux F, Morikawa Y, Cserjesi P and Gershon MD

    Department of Pathology and Cell Biology, Columbia University, P&S, New York, NY 10032, USA.

    Hand genes encode basic helix-loop-helix transcription factors that are expressed in the developing gut, where their function is unknown. We now report that enteric Hand2 expression is limited to crest-derived cells, whereas Hand1 expression is restricted to muscle and interstitial cells of Cajal. Hand2 is developmentally regulated and is intranuclear in precursors but cytoplasmic in neurons. Neurons develop in explants from wild-type but not Hand2(-/-) bowel, although, in both, crest-derived cells are present and glia arise. Similarly, small interfering RNA (siRNA) silencing of Hand2 in enteric crest-derived cells prevents neuronal development. Terminally differentiated enteric neurons do not develop after conditional inactivation of Hand2 in migrating crest-derived cells; nevertheless, conditional Hand2 inactivation does not prevent precursors from expressing early neural markers. We suggest that enteric neuronal development occurs in stages and that Hand2 expression is required for terminal differentiation but not for precursors to enter the neuronal lineage.

    Funded by: NINDS NIH HHS: NS12969, NS15547

    Development (Cambridge, England) 2007;134;12;2237-49

  • Zfp423/OAZ participates in a developmental switch during olfactory neurogenesis.

    Cheng LE and Reed RR

    Department of Molecular Biology and Genetics, Center for Sensory Biology, Johns Hopkins University School of Medicine, 725 N Wolfe Street, Baltimore, MD 21205, USA.

    The coordination of gene expression is critical for cell differentiation and the subsequent establishment of tissue function. We show here that a multiple zinc finger transcription factor, Zfp423/OAZ, is transiently expressed in newly differentiating olfactory-receptor neurons (ORNs) and has a key role in coordinating the expression of immature and mature stage-specific genes. OAZ deletion in mice impairs aspects of ORN differentiation, particularly the patterns of axonal projection to the olfactory bulb. OAZ gain-of-function experiments show that sustained OAZ expression throughout ORN maturation arrests ORN development at an immature stage and alters OR gene expression. Importantly, reintroducing OAZ expression in mature ORNs suppresses mature marker expression and reactivates immature-specific markers. Together, these experiments suggest that OAZ participates in a developmental switch regulating the transition from differentiation to maturation in ORNs.

    Funded by: NIDCD NIH HHS: R01 DC008295, R01 DC008295-02

    Neuron 2007;54;4;547-57

  • Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain.

    Zembrzycki A, Griesel G, Stoykova A and Mansouri A

    Max Planck Institute of Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg, 37077 Goettingen, Germany. azembrz@gwdg.de

    Background: The forebrain consists of multiple structures necessary to achieve elaborate functions. Proper patterning is, therefore, a prerequisite for the generation of optimal functional areas. Only a few factors have been shown to control the genetic networks that establish early forebrain patterning.

    Using conditional inactivation, we show that the transcription factor Sp8 has an essential role in the molecular and functional patterning of the developing telencephalon along the anteroposterior axis by modulating the expression gradients of Emx2 and Pax6. Moreover, Sp8 is essential for the maintenance of ventral cell identity in the septum and medial ganglionic eminence (MGE). This is probably mediated through a positive regulatory interaction with Fgf8 in the medial wall, and Nkx2.1 in the rostral MGE anlage, and independent of SHH and WNT signaling. Furthermore, Sp8 is required during corticogenesis to sustain a normal progenitor pool, and to control preplate splitting, as well as the specification of cellular diversity within distinct cortical layers.

    Neural development 2007;2;8

  • Differential impact of Lhx2 deficiency on expression of class I and class II odorant receptor genes in mouse.

    Hirota J, Omura M and Mombaerts P

    The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA; Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.

    Odorant receptor (OR) genes can be classified into two types: fish-like class I OR genes and mammalian-specific class II OR genes. We have previously shown that Lhx2, a LIM-homeodomain protein, binds to the homeodomain site in the promoter region of mouse M71, a class II OR, and that a knockout mutation in Lhx2 precludes expression of all tested class II OR genes including M71. Here, we report that most class I OR genes, which are expressed in a dorsal region of the olfactory epithelium, are still expressed in Lhx2-deficient embryos. There are two exceptions: two class I OR genes, which are normally expressed in a more ventral region, are no longer expressed in Lhx2 mutant mice. Lhx2 is transcribed in olfactory sensory neurons irrespective of expression of class I or class II OR genes. Thus, a deficiency of Lhx2 has a differential impact on class I and class II OR gene expression.

    Molecular and cellular neurosciences 2007;34;4;679-88

  • Identification of genes regulated by transcription factor KLF7 in differentiating olfactory sensory neurons.

    Kajimura D, Dragomir C, Ramirez F and Laub F

    Child Health Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, 89 French Street, New Brunswick, NJ 08901, United States.

    Gene targeting in mice has recently demonstrated that transcription factor KLF7 plays a critical role in neurite outgrowth and neuronal survival. Here we extended this genetic evidence by establishing the transcriptional profile of differentiating olfactory sensory neurons (OSNs) in Klf7(-/-) mice, and by identifying relevant genes that are directly regulated by KLF7. Functional clustering of DNA microarray data revealed that loss of KLF7 affects primarily the activity of genes involved in OSN differentiation, axonal growth, cytoskeletal dynamics, cell adhesion and synaptogenesis. Cell transfection experiments, on the other hand, demonstrated that the promoters of the genes encoding the OSN-specific OMP and the adhesion molecule L1 are both activated by KLF7 binding to CACCC motifs. Collectively, these results advance knowledge of transcriptional regulation of olfactory neurogenesis and KLF7 action.

    Funded by: NIAMS NIH HHS: AR38648

    Gene 2007;388;1-2;34-42

  • Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations.

    Munton RP, Tweedie-Cullen R, Livingstone-Zatchej M, Weinandy F, Waidelich M, Longo D, Gehrig P, Potthast F, Rutishauser D, Gerrits B, Panse C, Schlapbach R and Mansuy IM

    Brain Research Institute, Medical Faculty of the University of Zürich, Switzerland.

    Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

    Molecular & cellular proteomics : MCP 2007;6;2;283-93

  • Temporal and spatial expression profiles of the Fat3 protein, a giant cadherin molecule, during mouse development.

    Nagae S, Tanoue T and Takeichi M

    Graduate School of Biostudies, Kyoto University, Kyoto, Japan.

    Cadherins constitute a superfamily of cell-cell interaction molecules that participate in morphogenetic processes of animal development. Fat cadherins are the largest members of this superfamily, with 34 extracellular cadherin repeats. Classic Fat, identified in Drosophila, is known to regulate cell proliferation and planar cell polarity. Although 4 subtypes of Fat cadherin, Fat1, Fat2, Fat3, and Fat4/Fat-J, have been identified in vertebrates, their protein localization remains largely unknown. Here we describe the mRNA and protein distributions of Fat3 during mouse development. We found that Fat3 expression was restricted to the nervous system. In the brain, Fat3 was expressed in a variety of regions and axon fascicles. However, its strongest expression was observed in the olfactory bulb and retina. Detailed analysis of Fat3 in the developing olfactory bulb revealed that Fat3 mRNA was mainly expressed by mitral cells and that its proteins were densely localized along the dendrites of these cells as well as in their axons to some extent. Fat3 transcripts in the retina were expressed by amacrine and ganglion cells, and its proteins were concentrated in the inner plexiform layer throughout development. Based on these observations, we suggest that Fat3 plays a role in the interactions between neurites derived from specific subsets of neurons during development.

    Developmental dynamics : an official publication of the American Association of Anatomists 2007;236;2;534-43

  • The Rho-GTPase cdc42 regulates neural progenitor fate at the apical surface.

    Cappello S, Attardo A, Wu X, Iwasato T, Itohara S, Wilsch-Bräuninger M, Eilken HM, Rieger MA, Schroeder TT, Huttner WB, Brakebusch C and Götz M

    GSF, National Research Center for Environment and Health, Institute for Stem Cell Research, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Munich, Germany.

    Stem cell persistence into adulthood requires self-renewal from early developmental stages. In the developing mouse brain, only apical progenitors located at the ventricle are self-renewing, whereas basal progenitors gradually deplete. However, nothing is known about the mechanisms regulating the fundamental difference between these progenitors. Here we show that the conditional deletion of the small Rho-GTPase cdc42 at different stages of neurogenesis in mouse telencephalon results in an immediate increase in basal mitoses. Whereas cdc42-deficient progenitors have normal cell cycle length, orientation of cell division and basement membrane contact, the apical location of the Par complex and adherens junctions are gradually lost, leading to an increasing failure of apically directed interkinetic nuclear migration. These cells then undergo mitoses at basal positions and acquire the fate of basal progenitors. Thus, cdc42 has a crucial role at the apical pole of progenitors, thereby regulating the position of mitoses and cell fate.

    Nature neuroscience 2006;9;9;1099-107

  • From the Cover: Indispensability of the glutamate transporters GLAST and GLT1 to brain development.

    Matsugami TR, Tanemura K, Mieda M, Nakatomi R, Yamada K, Kondo T, Ogawa M, Obata K, Watanabe M, Hashikawa T and Tanaka K

    Laboratory of Molecular Neuroscience, School of Biomedical Science and Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan.

    Previous in vitro studies have shown that the neurotransmitter glutamate is important in brain development. Paradoxically, loss-of-function mouse models of glutamatergic signaling that are generated by genetic deletion of glutamate receptors or glutamate release show normal brain assembly. We examined the direct consequences on brain development of extracellular glutamate buildup due to the depletion of the glutamate transporters GLAST and GLT1. GLAST/GLT1 double knockout mice show multiple brain defects, including cortical, hippocampal, and olfactory bulb disorganization with perinatal mortality. Here, we report abnormal formation of the neocortex in GLAST/GLT1 mutants. Several essential aspects of neuronal development, such as stem cell proliferation, radial migration, neuronal differentiation, and survival of SP neurons, were impaired. These results provide direct in vivo evidence that GLAST and GLT1 are necessary for brain development through regulation of extracellular glutamate concentration and show that an important mechanism is likely to be maintenance of glutamate-mediated synaptic transmission.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;32;12161-6

  • Mice without transcription factor KLF7 provide new insight into olfactory bulb development.

    Laub F, Dragomir C and Ramirez F

    Child Health Institute of New Jersey-UMDNJ-Robert W. Johnson Medical School, 89 French Street, New Brunswick, NJ 08901, USA.

    Recent genetic studies have excluded that peripheral innervation plays a substantial role in the initial outgrowth of the olfactory bulb. Mice without Kruppel-like factor 7 activity die at birth and display hypoplastic olfactory bulbs which lack peripheral innervation. Here, we report that incomplete penetrance of the mutation is responsible for partial bulb innervation in a small fraction of Klf7 null mice. Analysis of the partially innervated bulbs of mutant embryos, newborns and adult mice revealed an obligatory correlation with local restoration of laminar architecture, neuronal cell differentiation and neuronal activity. The degree of normal OB maturation in Klf7-/- OBs was proportional to the degree of peripheral innervation. These findings therefore indicate that peripheral innervation contributes to bulb maturation late in development by promoting cell morphogenesis and differentiation.

    Funded by: NIAMS NIH HHS: AR38648

    Brain research 2006;1103;1;108-13

  • The docking protein Cas links tyrosine phosphorylation signaling to elongation of cerebellar granule cell axons.

    Huang J, Sakai R and Furuichi T

    Laboratory for Molecular Neurogenesis, Riken Brain Science Institute, Wako, Saitama 351-0198, USA.

    Crk-associated substrate (Cas) is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of the actin cytoskeletal organization and cell migration in fibroblasts. The function of Cas in neurons, however, is poorly understood. Here we report that Cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. During cerebellar development, Cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. Cas coimmunoprecipitates with Src family protein tyrosine kinases, Crk, and cell adhesion molecules and colocalizes with these proteins in granule cells. The axon extension of granule cells is inhibited by either RNA interference knockdown of Cas or overexpression of the Cas mutant lacking the YDxP motifs, which are tyrosine phosphorylated and thereby interact with Crk. These findings demonstrate that Cas acts as a key scaffold that links the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation.

    Molecular biology of the cell 2006;17;7;3187-96

  • Expression and distribution of JNK/SAPK-associated scaffold protein JSAP1 in developing and adult mouse brain.

    Miura E, Fukaya M, Sato T, Sugihara K, Asano M, Yoshioka K and Watanabe M

    Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan.

    The c-Jun N-terminal kinase (JNK) is one of the three major mitogen-activated protein kinases (MAPKs) playing key roles in various cellular processes in response to both extracellular and intracellular stimuli. JNK/SAPK-associated protein 1 (JSAP1 also referred to as JIP3) is a JNK-associated scaffold that controls the specificity and efficiency of JNK signaling cascades. Here we studied its expression in mouse brains. JSAP1 mRNA was expressed in developing and adult brains, showing spatial patterns similar to JNK1-3 mRNAs. In embryos, JSAP1 immunolabeling was intense for progenitor cells in the ventricular zone throughout the brain and in the external granular layer of the cerebellum, and for neurons and glial cells differentiating in the mantle zone. In adults, JSAP1 was distributed in various neurons and Bergmann glia, with higher levels in striatal cholinergic interneurons, telencephalic parvalbumin-positive interneurons and cerebellar Purkinje cells. In these neurons, JSAP1 was observed as tiny particulate staining in spines, dendrites, perikarya and axons, where it was often associated with the smooth endoplasmic reticulum (sER) and cell membrane. Immunoblots revealed enriched distribution in the microsomal fraction and cytosolic fraction. Therefore, the characteristic cellular expression and subcellular distribution of JSAP1 might be beneficial for cells to efficiently link external stimuli to the JNK MAPK pathway and other intracellular machineries.

    Journal of neurochemistry 2006;97;5;1431-46

  • Identification of cis-acting regions that contribute to neuron-specific expression of the GAP-43 gene.

    Takahashi M, Sato Y, Nakagami Y, Miyake K and Iijima S

    Department of Biotechnology, Graduate School of Engieering, Nagoya University.

    There are two transcription start sites in the growth-associated protein 43 (GAP-43) promoter, and several repressive elements have been reported in the control region. But the repressive effects have been analyzed only for the distal transcription start site. Among the repressive elements reported, we found that modulator I repressed GAP-43 gene expression from the proximal promoter in non-neuronal cells. We also found a novel stimulative element immediately downstream of modulator I.

    Bioscience, biotechnology, and biochemistry 2006;70;6;1492-5

  • Midline radial glia translocation and corpus callosum formation require FGF signaling.

    Smith KM, Ohkubo Y, Maragnoli ME, Rasin MR, Schwartz ML, Sestan N and Vaccarino FM

    Child Study Center, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

    Midline astroglia in the cerebral cortex develop earlier than other astrocytes through mechanisms that are still unknown. We show that radial glia in dorsomedial cortex retract their apical endfeet at midneurogenesis and translocate to the overlaying pia, forming the indusium griseum. These cells require the fibroblast growth factor receptor 1 (Fgfr1) gene for their precocious somal translocation to the dorsal midline, as demonstrated by inactivating the Fgfr1 gene in radial glial cells and by RNAi knockdown of Fgfr1 in vivo. Dysfunctional astroglial migration underlies the callosal dysgenesis in conditional Fgfr1 knockout mice, suggesting that precise targeting of astroglia to the cortex has unexpected roles in axon guidance. FGF signaling is sufficient to induce somal translocation of radial glial cells throughout the cortex; furthermore, the targeting of astroglia to dorsolateral cortex requires FGFr2 signaling after neurogenesis. Hence, FGFs have an important role in the transition from radial glia to astrocytes by stimulating somal translocation of radial glial cells.

    Funded by: NICHD NIH HHS: HD045481; NIMH NIH HHS: 5T32-MH18268, MH067715; NINDS NIH HHS: NS054273, NS35476

    Nature neuroscience 2006;9;6;787-97

  • Compensation by tumor suppressor genes during retinal development in mice and humans.

    Donovan SL, Schweers B, Martins R, Johnson D and Dyer MA

    Department of Developmental Neurobiology, St, Jude Children's Research Hospital, Memphis, TN 38105, USA. stacy.donovan@stjude.org

    Background: The RB1 gene was the first tumor suppressor gene cloned from humans by studying genetic lesions in families with retinoblastoma. Children who inherit one defective copy of the RB1 gene have an increased susceptibility to retinoblastoma. Several years after the identification of the human RB1 gene, a targeted deletion of Rb was generated in mice. Mice with one defective copy of the Rb gene do not develop retinoblastoma. In this manuscript, we explore the different roles of the Rb family in human and mouse retinal development in order to better understand the species-specific difference in retinoblastoma susceptibility.

    Results: We found that the Rb family of proteins (Rb, p107 and p130) are expressed in a dynamic manner during mouse retinal development. The primary Rb family member expressed in proliferating embryonic retinal progenitor cells in mice is p107, which is required for appropriate cell cycle exit during retinogenesis. The primary Rb family member expressed in proliferating postnatal retinal progenitor cells is Rb. p130 protein is expressed redundantly with Rb in postmitotic cells of the inner nuclear layer and the ganglion cell layer of the mouse retina. When Rb is inactivated in an acute or chronic manner during mouse retinal development, p107 is upregulated in a compensatory manner. Similarly, when p107 is inactivated in the mouse retina, Rb is upregulated. No changes in p130 expression were seen when p107, Rb or both were inactivated in the developing mouse retina. In the human retina, RB1 was the primary family member expressed throughout development. There was very little if any p107 expressed in the developing human retina. In contrast to the developing mouse retina, when RB1 was acutely inactivated in the developing human fetal retina, p107 was not upregulated in a compensatory manner.

    Conclusion: We propose that intrinsic genetic compensation between Rb and p107 prevents retinoblastoma in Rb- or p107-deficient mice, but this compensation does not occur in humans. Together, these data suggest a model that explains why humans are susceptible to retinoblastoma following RB1 loss, but mice require both Rb and p107 gene inactivation.

    BMC biology 2006;4;14

  • Expression profiling the developing mammalian enteric nervous system identifies marker and candidate Hirschsprung disease genes.

    Heanue TA and Pachnis V

    Division of Molecular Neurobiology, National Institute for Medical Research, Medical Research Council, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.

    The enteric nervous system (ENS) is composed of neurons and glial cells, organized as interconnected ganglia within the gut wall, which controls peristalsis of the gut wall and secretions from its glands. The Ret receptor tyrosine kinase is expressed throughout enteric neurogenesis and is required for normal ENS development; humans with mutations in the RET locus have Hirschsprung disease (HSCR, an absence of ganglia in the colon), and mice lacking Ret have total intestinal aganglionosis. The Ret mutant mouse provides a tool for identifying genes implicated in development of the ENS. By using RNA from WT and Ret mutant (aganglionic) gut tissue and DNA microarrays, we have conducted a differential screen for ENS-expressed genes and have identified hundreds of candidate ENS-expressed genes. Forty-seven genes were selected for further analysis, representing diverse functional classes. We show that all of the analyzed genes are expressed in the ENS and that the screen was sensitive enough to identify genes marking only subpopulations of ENS cells. Our screen, therefore, was reliable and sensitive and has identified many previously undescribed genes for studying ENS development. Moreover, two of the genes identified in our screen Arhgef3 and Ctnnal1, have human homologues that map to previously identified HSCR susceptibility loci, thus representing excellent candidates for HSCR genes. This comprehensive profile of ENS gene expression refines our understanding of ENS development and serves as a resource for future developmental, biochemical, and human genetic studies.

    Funded by: Medical Research Council: MC_U117537087; NCI NIH HHS: CA23767, P01 CA023767

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;18;6919-24

  • Comprehensive identification of phosphorylation sites in postsynaptic density preparations.

    Trinidad JC, Specht CG, Thalhammer A, Schoepfer R and Burlingame AL

    Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA.

    In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and approximately 80% of identified phosphorylation sites are novel.

    Funded by: NCRR NIH HHS: RR14606; Wellcome Trust

    Molecular & cellular proteomics : MCP 2006;5;5;914-22

  • BGEM: an in situ hybridization database of gene expression in the embryonic and adult mouse nervous system.

    Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T, Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States.

    Funded by: NINDS NIH HHS: 5R37NS036558, N01-NS-0-2331, R37 NS036558

    PLoS biology 2006;4;4;e86

  • Gene expression analysis identifies novel genes participating in early murine liver development and adult liver regeneration.

    Jochheim-Richter A, Rüdrich U, Koczan D, Hillemann T, Tewes S, Petry M, Kispert A, Sharma AD, Attaran F, Manns MP and Ott M

    Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany.

    Adult liver tissue regeneration may recapitulate molecular events of liver organogenesis. As gaps in our understanding of the fundamental processes that govern development and regeneration of the liver still exist, we studied gene expression in the developing liver at embryonic day 9.5 post coitum (E d9.5 p.c.). Microarray data from E d9.5 p.c. as well as previously published data from embryonic day 11.5 post coitum (E d11.5 p.c.) and embryonic day 13.5 post coitum (E d13.5 p.c.) were subjected to cluster analysis. This led to the identification of 130 genes which were characterized by continuous expression at all stages of liver development with peak expression of 44 genes at E d9.5 p.c. Five of these genes, previously not known to be associated with early liver development or with adult liver regeneration were selected for further analysis. The expression of the genes was studied by real-time polymerase chain reaction at 0, 2, 4, 6, 12, 24 and 48 hr after partial hepatectomy in the adult liver. Two of the genes, growth arrest protein 43 (GAP43) and paired-like homeodomain transcription factor 2 (Pitx2) were exclusively detected at 24 hr, whereas the genes Twist1, Midkine, and zinc finger protein of cerebellum 1 (Zic1) each showed a specific expression profile in the regenerating liver with peak expressions at 4, 24, and 6 hr, respectively. In summary, we were able to identify novel genes, that may act as regulators during liver formation as well as in the regeneration phase of adult liver. This information may contribute to the development of new targets for the treatment of liver diseases in the future.

    Differentiation; research in biological diversity 2006;74;4;167-73

  • Zinc-finger gene Fez in the olfactory sensory neurons regulates development of the olfactory bulb non-cell-autonomously.

    Hirata T, Nakazawa M, Yoshihara S, Miyachi H, Kitamura K, Yoshihara Y and Hibi M

    Laboratory for Vertebrate Axis Formation, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan.

    Fez is a zinc-finger gene encoding a transcriptional repressor that is expressed in the olfactory epithelium, hypothalamus, ventrolateral pallium and prethalamus at mid-gestation. To reveal its function, we generated Fez-deficient mice. The Fez-deficient mice showed several abnormalities in the olfactory system: (1) impaired axonal projection of the olfactory sensory neurons; (2) reduced size of the olfactory bulb; (3) abnormal layer formation in the olfactory bulb; and (4) aberrant rostral migration of the interneuron progenitors. Fez was not expressed in the projection neurons, interneurons or interneuron progenitors. Transgene-mediated expression of Fez in olfactory sensory neurons significantly rescued the abnormalities in olfactory axon projection and in the morphogenesis of the olfactory bulb in Fez-knockout mice. Thus, Fez is cell-autonomously required for the axon termination of olfactory sensory neurons, and Fez non-cell-autonomously controls layer formation and interneuron development in the olfactory bulb. These findings suggest that signals from olfactory sensory neurons contribute to the proper formation of the olfactory bulb.

    Development (Cambridge, England) 2006;133;8;1433-43

  • Notch 1 inhibits photoreceptor production in the developing mammalian retina.

    Jadhav AP, Mason HA and Cepko CL

    Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

    The transmembrane receptor Notch1 plays a role in development and homeostasis in vertebrates and invertebrates. The mammalian retina is an excellent tissue in which to dissect the precise role of Notch signaling in regulating cell fate and proliferation. However, a systematic analysis has been limited by the early embryonic lethality of Notch1-null mice. Here, Notch1 was conditionally removed from the murine retina either early or late in development. Removal of Notch1 early led to a reduction in the size of the retina as well as aberrant morphology. A decrease in the number of progenitor cells and premature neurogenesis accounted for the reduction in size. Unexpectedly, ablation of Notch1 in early progenitor cells led to enhanced cone photoreceptor production, and ablation of Notch1 at later points led to an almost exclusive production of rod photoreceptor cells. These data suggest that Notch1 not only maintains the progenitor state, but is required to inhibit the photoreceptor fate. These cone enriched mutant mice should prove to be a valuable resource for the study of this relatively rare mammalian photoreceptor cell type.

    Funded by: NEI NIH HHS: T32EY007110; PHS HHS: EYO 86767

    Development (Cambridge, England) 2006;133;5;913-23

  • Anomalous functional organization of barrel cortex in GAP-43 deficient mice.

    Dubroff JG, Stevens RT, Hitt J, Hodge CJ and McCasland JS

    Graduate Program in Neuroscience, SUNY Upstate Medical University, Syracuse, NY 13210, USA. dubroffj@upstate.edu

    Growth associated protein 43 (GAP-43), found only in the nervous system, regulates the response of neurons to axon guidance signals. It is also critical for establishing normal somatotopy. Mice lacking GAP-43 (KO) show aberrant pathfinding by thalamocortical afferents, and do not form cortical whisker/barrels. GAP-43 heterozygous (HZ) mice show more subtle deficits--delayed barrel segregation and enlarged barrels at postnatal day 7. Here, we used cortical intrinsic signal imaging to characterize adult somatotopy in wildtype (WT), GAP-43 KO, and HZ mice. We found clear foci of activation in GAP-43 KO cortex in response to single-whisker stimulation. However, the KO spatial activation patterns showed severe anomalies, indicating a loss of functional somatotopy. In some cases, multiple foci were activated by single whiskers, while in other cases, the same cortical zone was activated by several whiskers. The results are consistent with our previous findings of aberrant pathfinding and clustering by thalamocortical afferent axons, and absence of barrel patterning. Our findings indicate that cortex acts to cluster afferents from a given whisker, even in the absence of normal topography. By contrast, single-whisker stimulation revealed normal adult topographic organization in WT and HZ mice. However, we found that functional representations of adult HZ barrels are larger than those found in WT mice. Since histological HZ barrels recover normal dimensions by postnatal day 26, the altered circuit function in GAP-43 HZ cortex could be a secondary consequence of the rescue of barrel dimensions.

    Funded by: NICHD NIH HHS: HD04483101; NINDS NIH HHS: NS31829, NS40779

    NeuroImage 2006;29;4;1040-8

  • Links between alpha-catenin, NF-kappaB, and squamous cell carcinoma in skin.

    Kobielak A and Fuchs E

    Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY 10021, USA.

    Cancers display a diverse set of cellular defects, which are thought to be elicited by multiple genetic mutations. In this study, we show that when a single adherens junction protein, alpha-catenin, is removed by conditional targeting, the entire skin epidermis systematically transforms to a hyperproliferative, invasive tissue replete with inflammation. Transcriptional profiling and biochemical analyses reveal that alpha-catenin ablation is accompanied by activation of NF-kappaB and its proinflammatory target genes, along with genes involved in proliferation, wound healing, angiogenesis, and metastasis. Many of these alterations occur in vitro and in the embryo, and thus seem at least partly to be intrinsic to the loss of alpha-catenin. We show that reductions in alpha-catenin, activation of NF-kappaB, and inflammation are common features of human squamous cell carcinomas of the skin.

    Funded by: NIAMS NIH HHS: AR27883, R01 AR027883, R37 AR027883

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;7;2322-7

  • In vivo post-transcriptional regulation of GAP-43 mRNA by overexpression of the RNA-binding protein HuD.

    Bolognani F, Tanner DC, Merhege M, Deschênes-Furry J, Jasmin B and Perrone-Bizzozero NI

    Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.

    HuD is a neuronal-specific RNA-binding protein that binds to and stabilizes the mRNAs of growth-associated protein-43 (GAP-43) and other neuronal proteins. HuD expression increases during brain development, nerve regeneration, and learning and memory, suggesting that this protein is important for controlling gene expression during developmental and adult plasticity. To examine the function of HuD in vivo, we generated transgenic mice overexpressing human HuD under the control of the calcium-calmodulin-dependent protein kinase IIalpha promoter. The transgene was expressed at high levels throughout the forebrain, including the hippocampal formation, amygdala and cerebral cortex. Using quantitative in situ hybridization, we found that HuD overexpression led to selective increases in GAP-43 mRNA in hippocampal dentate granule cells and neurons in the lateral amygdala and layer V of the neorcortex. In contrast, GAP-43 pre-mRNA levels were unchanged or decreased in the same neuronal populations. Comparison of the levels of mature GAP-43 mRNA and pre-mRNA in the same neurons of transgenic mice suggested that HuD increased the stability of the transcript. Confirming this, mRNA decay assays revealed that the GAP-43 mRNA was more stable in brain extracts from HuD transgenic mice than non-transgenic littermates. In conclusion, our results demonstrate that HuD overexpression is sufficient to increase GAP-43 mRNA stability in vivo.

    Funded by: NIAAA NIH HHS: T32 AA1427; NINDS NIH HHS: NS30255

    Journal of neurochemistry 2006;96;3;790-801

  • Multiple Eph receptors and B-class ephrins regulate midline crossing of corpus callosum fibers in the developing mouse forebrain.

    Mendes SW, Henkemeyer M and Liebl DJ

    Neuroscience Program, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.

    Agenesis of the corpus callosum (CC) is a rare birth defect that occurs in isolated conditions and in combination with other developmental cerebral abnormalities. Recent identification of families of growth and guidance molecules has generated interest in the mechanisms that regulate callosal growth. One family, ephrins and Eph receptors, has been implicated in mediating midline pathfinding decisions; however, the complexity of these interactions has yet to be unraveled. Our studies shed light on which B-class ephrins and Eph receptors function to regulate CC midline growth and how these molecules interact with important guideposts during development. We show that multiple Eph receptors (B1, B2, B3, and A4) and B-class ephrins (B1, B2, and B3) are present and function in developing forebrain callosal fibers based on both spatial and temporal expression patterns and analysis of gene-targeted knock-out mice. Defects are most pronounced in the combination double knock-out mice, suggesting that compensatory mechanisms exist for several of these family members. Furthermore, these CC defects range from mild hypoplasia to complete agenesis and Probst's bundle formation. Further analysis revealed that Probst's bundle formation may reflect aberrant glial formations and/or altered sensitivity of CC axons to other guidance cues. Our results support a significant role for ephrins and Eph receptors in CC development and may provide insight to possible mechanisms involved in axon midline crossing and human disorder.

    Funded by: NIMH NIH HHS: MH66332, R01 MH066332; NINDS NIH HHS: NS30291

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;3;882-92

  • Neurogranin expression identifies a novel array of Purkinje cell parasagittal stripes during mouse cerebellar development.

    Larouche M, Che PM and Hawkes R

    Department of Cell Biology and Anatomy, Genes and Development Research Group, Hotchkiss Brain Institute, Faculty of Medicine, The University of Calgary, Alberta T2N 4N1, Canada.

    Markers that reveal the parasagittal organization of cerebellar Purkinje cells may be grouped into two classes based on the time during development when they are expressed. In mice, early-onset markers are defined by their heterogeneous expression in clusters of Purkinje cells during late embryogenesis, which disappears shortly following birth. Late-onset markers are generally not expressed until about 1 week after birth and do not reach a stable striped expression pattern until about 3 weeks postnatally. Currently, no endogenous markers are known that are heterogeneously expressed in the temporal gap between these two classes. Here we present immunocytochemical evidence that parasagittal stripes of Purkinje cells express a member of the calpacitin protein family, neurogranin, possibly from as early as embryonic day (E) 13 and definitively from E15, in a pattern that persists up to postnatal day (P) 20. Neurogranin is thus the first endogenous marker of a Purkinje cell subset capable of bridging the temporal gap between the early- and late-onset patterns. In the early neonate, up to five pairs of neurogranin-immunopositive Purkinje cell stripes run parasagittally through the cerebellum, with the exact number dependent on the rostrocaudal position. Expression is lost during postnatal development in a transverse zone-dependent fashion. Purkinje cells in the central and nodular zones lose neurogranin expression between approximately P4 and P6, whereas expression in the posterior zone persists until approximately P20. Neurogranin immunoreactivity will be a valuable tool in helping to clarify the relationships between early- and late-onset patterns.

    The Journal of comparative neurology 2006;494;2;215-27

  • Development of midline cell types and commissural axon tracts requires Fgfr1 in the cerebrum.

    Tole S, Gutin G, Bhatnagar L, Remedios R and Hébert JM

    Department of Biological Sciences, Room B304, Tata Institute of Fundamental Research, Colaba, Mumbai, India. stole@tifr.res.in

    The adult cerebral hemispheres are connected to each other by specialized midline cell types and by three axonal tracts: the corpus callosum, the hippocampal commissure, and the anterior commissure. Many steps are required for these tracts to form, including early patterning and later axon pathfinding steps. Here, the requirement for FGF signaling in forming midline cell types and commissural axon tracts of the cerebral hemispheres is examined. Fgfr1, but not Fgfr3, is found to be essential for establishing all three commissural tracts. In an Fgfr1 mutant, commissural neurons are present and initially project their axons, but these fail to cross the midline that separates the hemispheres. Moreover, midline patterning defects are observed in the mutant. These defects include the loss of the septum and three specialized glial cell types, the indusium griseum glia, midline zipper glia, and glial wedge. Our findings demonstrate that FGF signaling is required for generating telencephalic midline structures, in particular septal and glial cell types and all three cerebral commissures. In addition, analysis of the Fgfr1 heterozygous mutant, in which midline patterning is normal but commissural defects still occur, suggests that at least two distinct FGF-dependent mechanisms underlie the formation of the cerebral commissures.

    Funded by: NIGMS NIH HHS: T32 GM007288

    Developmental biology 2006;289;1;141-51

  • Math1 target genes are enriched with evolutionarily conserved clustered E-box binding sites.

    Krizhanovsky V, Soreq L, Kliminski V and Ben-Arie N

    Department of Cell and Animal Biology, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.

    The basic helix-loop-helix (bHLH) transcription factor Math1 and its orthologs are fundamental for proper development of various neuronal subpopulations, such as cerebellar granule cells, D1 interneurons in the spinal cord, and inner ear hair cells. Although crucial for neurogenesis, the mechanisms by which Math1 specifically recognizes its direct targets are not fully understood. To search for direct and indirect target genes and signaling pathways controlled by Math1, we analyzed the effect of Math1 knockout on the expression profile of multiple genes in the embryonic cerebellum. Eighteen differentially expressed transcripts were identified and found to belong to a few developmentally-related functional groups, such as transcriptional regulation, proliferation, organogenesis, signal transduction, and apoptosis. Importantly, genomic analysis of E-box motifs has identified a significant enrichment and clustering of MATH1-binding E-boxes only in a subset of differentially expressed genes (Nr2f6, Hras1, and Hes5) in both mouse and man. Moreover, Math1 was shown by chromatin immunoprecipitation (ChIP) to bind, and by a luciferase reporter assay to activate transcription, of an upstream genomic fragment of Nr2f6. Taken together, we propose that when putative direct targets of Math1 are being selected for detailed studies on DNA microarray hybridization, the enrichment and clustering of binding E-boxes in multiple species may be helpful criteria. Our findings may be useful to the study of other bHLH transcription factors, many of which control the development of the nervous system.

    Journal of molecular neuroscience : MN 2006;28;2;211-29

  • Stage-specific induction of DNA methyltransferases in olfactory receptor neuron development.

    MacDonald JL, Gin CS and Roskams AJ

    Department of Zoology, University of British Columbia, 6270 University Boulevard, Rm 3479, Vancouver, B.C., Canada V6T 1Z4.

    DNA methylation-dependent gene silencing, mediated by DNA methyltransferases (DNMTs), is essential for normal mammalian development and its dysregulation has been implicated in neurodevelopmental disorders. Despite this, little is known about DNMTs in the developing or mature nervous system. Here, we show that DNMT1, 3a and 3b are expressed at discrete developmental stages in the olfactory neuron lineage, coincident with key shifts in developmental gene expression. DNMT1 is induced in cycling progenitors and is retained in post-mitotic olfactory receptor neurons (ORNs). DNMT3b is restricted to mitotic olfactory progenitors, whereas DNMT3a is expressed only in post-mitotic immature neurons prior to ORN terminal maturation, coincident with histone deacetylase 2 (HDAC2), a key downstream effector of methylation-dependent chromatin condensation. Similar stage-specific expression of DNMT3b and 3a was also found in other developing sensory and CNS neurons. This suggests that progressive lineage restriction regulated by methylation-dependent silencing could be a highly conserved mechanism shared by multiple lineages in the developing nervous system.

    Developmental biology 2005;288;2;461-73

  • Two quantitative trait loci for prepulse inhibition of startle identified on mouse chromosome 16 using chromosome substitution strains.

    Petryshen TL, Kirby A, Hammer RP, Purcell S, O'Leary SB, Singer JB, Hill AE, Nadeau JH, Daly MJ and Sklar P

    Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Broad Institute of Harvard, 185 Cambridge Street, Cambridge, MA 02139, USA.

    Prepulse inhibition (PPI) of acoustic startle is a genetically complex quantitative phenotype of considerable medical interest due to its impairment in psychiatric disorders such as schizophrenia. To identify quantitative trait loci (QTL) involved in mouse PPI, we studied mouse chromosome substitution strains (CSS) that each carry a homologous chromosome pair from the A/J inbred strain on a host C57BL/6J inbred strain background. We determined that the chromosome 16 substitution strain has elevated PPI compared to C57BL/6J (P = 1.6 x 10(-11)), indicating that chromosome 16 carries one or more PPI genes. QTL mapping using 87 F(2) intercross progeny identified two significant chromosome 16 loci with LODs of 3.9 and 4.7 (significance threshold LOD is 2.3). The QTL were each highly significant independently and do not appear to interact. Sequence variation between B6 and A/J was used to identify strong candidate genes in the QTL regions, some of which have known neuronal functions. In conclusion, we used mouse CSS to rapidly and efficiently identify two significant QTL for PPI on mouse chromosome 16. The regions contain a limited number of strong biological candidate genes that are potential risk genes for psychiatric disorders in which patients have PPI impairments.

    Funded by: NCRR NIH HHS: P40 RR012305, RR 12305; NIMH NIH HHS: MH 066954, R01 MH066954, R21 MH071673

    Genetics 2005;171;4;1895-904

  • Molecular control of spinal accessory motor neuron/axon development in the mouse spinal cord.

    Dillon AK, Fujita SC, Matise MP, Jarjour AA, Kennedy TE, Kollmus H, Arnold HH, Weiner JA, Sanes JR and Kaprielian Z

    Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

    Within the developing vertebrate spinal cord, motor neuron subtypes are distinguished by the settling positions of their cell bodies, patterns of gene expression, and the paths their axons follow to exit the CNS. The inclusive set of cues required to guide a given motor axon subtype from cell body to target has yet to be identified, in any species. This is attributable, in part, to the unavailability of markers that demarcate the complete trajectory followed by a specific class of spinal motor axons. Most spinal motor neurons extend axons out of the CNS through ventral exit points. In contrast, spinal accessory motor neurons (SACMNs) project dorsally directed axons through lateral exit points (LEPs), and these axons assemble into the spinal accessory nerve (SAN). Here we show that an antibody against BEN/ALCAM/SC1/DM-GRASP/MuSC selectively labels mouse SACMNs and can be used to trace the pathfinding of SACMN axons. We use this marker, together with a battery of transcription factor-deficient or guidance cue/receptor-deficient mice to identify molecules required for distinct stages of SACMN development. Specifically, we find that Gli2 is required for the initial extension of axons from SACMN cell bodies, and that netrin-1 and its receptor Dcc are required for the proper dorsal migration of these cells and the dorsally directed extension of SACMN axons toward the LEPs. Furthermore, in the absence of the transcription factor Nkx2.9, SACMN axons fail to exit the CNS. Together, these findings suggest molecular mechanisms that are likely to regulate key steps in SACMN development.

    Funded by: NINDS NIH HHS: F31 NS43852-03

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;44;10119-30

  • Growth-associated protein GAP-43 and L1 act synergistically to promote regenerative growth of Purkinje cell axons in vivo.

    Zhang Y, Bo X, Schoepfer R, Holtmaat AJ, Verhaagen J, Emson PC, Lieberman AR and Anderson PN

    Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom. yi.zhang@qmul.ac.uk

    Neuronal expression of growth-associated protein 43 (GAP-43) and the cell adhesion molecule L1 has been correlated with CNS axonal growth and regeneration, but it is not known whether expression of these molecules is necessary for axonal regeneration to occur. We have taken advantage of the fact that Purkinje cells do not express GAP-43 or L1 in adult mammals or regenerate axons into peripheral nerve grafts to test the importance of these molecules for axonal regeneration in vivo. Transgenic mice were generated in which Purkinje cells constitutively express L1 or both L1 and GAP-43 under the Purkinje cell-specific L7 promoter, and regeneration of Purkinje cell axons into peripheral nerve grafts implanted into the cerebellum was examined. Purkinje cells expressing GAP-43 or L1 showed minor enhancement of axonal sprouting. Purkinje cells expressing both GAP-43 and L1 showed more extensive axonal sprouting and axonal growth into the proximal portion of the graft. When a predegenerated nerve graft was implanted into double-transgenic mice, penetration of the graft by Purkinje cell axonal sprouts was strongly enhanced, and some axons grew along the entire intracerebral length of the graft (2.5-3.0 mm) and persisted for several months. The results demonstrate that GAP-43 and L1 coexpressed in Purkinje cells can act synergistically to switch these regeneration-incompetent CNS neurons into a regeneration-competent phenotype and show that coexpression of these molecules is a key regulator of the regenerative ability of intrinsic CNS neurons in vivo.

    Funded by: Wellcome Trust

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;41;14883-8

  • Developmental abnormalities of neuronal structure and function in prenatal mice lacking the prader-willi syndrome gene necdin.

    Pagliardini S, Ren J, Wevrick R and Greer JJ

    Department of Physiology, Centre of Neuroscience, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada.

    Necdin (Ndn) is one of a cluster of genes deleted in the neurodevelopmental disorder Prader-Willi syndrome (PWS). Ndntm2Stw mutant mice die shortly after birth because of abnormal respiratory rhythmogenesis generated by a key medullary nucleus, the pre-Bötzinger complex (preBötC). Here, we address two fundamental issues relevant to its pathogenesis. First, we performed a detailed anatomical study of the developing medulla to determine whether there were defects within the preBötC or synaptic inputs that regulate respiratory rhythmogenesis. Second, in vitro studies determined if the unstable respiratory rhythm in Ndntm2Stw mice could be normalized by neuromodulators. Anatomical defects in Ndntm2Stw mice included defasciculation and irregular projections of axonal tracts, aberrant neuronal migration, and a major defect in the cytoarchitecture of the cuneate/gracile nuclei, including dystrophic axons. Exogenous application of neuromodulators alleviated the long periods of slow respiratory rhythms and apnea, but some instability of rhythmogenesis persisted. We conclude that deficiencies in the neuromodulatory drive necessary for preBötC function contribute to respiratory dysfunction of Ndntm2Stw mice. These abnormalities are part of a more widespread deficit in neuronal migration and the extension, arborization, and fasciculation of axons during early stages of central nervous system development that may account for respiratory, sensory, motor, and behavioral problems associated with PWS.

    The American journal of pathology 2005;167;1;175-91

  • Transcription factor KLF7 is important for neuronal morphogenesis in selected regions of the nervous system.

    Laub F, Lei L, Sumiyoshi H, Kajimura D, Dragomir C, Smaldone S, Puche AC, Petros TJ, Mason C, Parada LF and Ramirez F

    Laboratory of Genetics and Organogenesis, Research Division of the Hospital for Special Surgery, and Department of Physiology and Biophysics at Weill Medical College of Cornell University, 535 East 70th St., New York, New York 10021, USA.

    The Krüppel-like transcription factors (KLFs) are important regulators of cell proliferation and differentiation in several different organ systems. The mouse Klf7 gene is strongly active in postmitotic neuroblasts of the developing nervous system, and the corresponding protein stimulates transcription of the cyclin-dependent kinase inhibitor p21waf/cip gene. Here we report that loss of KLF7 activity in mice leads to neonatal lethality and a complex phenotype which is associated with deficits in neurite outgrowth and axonal misprojection at selected anatomical locations of the nervous system. Affected axon pathways include those of the olfactory and visual systems, the cerebral cortex, and the hippocampus. In situ hybridizations and immunoblots correlated loss of KLF7 activity in the olfactory epithelium with significant downregulation of the p21waf/cip and p27kip1 genes. Cotransfection experiments extended the last finding by documenting KLF7's ability to transactivate a reporter gene construct driven by the proximal promoter of p27kip1. Consistent with emerging evidence for a role of Cip/Kip proteins in cytoskeletal dynamics, we also documented p21waf/cip and p27kip1 accumulation in the cytoplasm of differentiating olfactory sensory neurons. KLF7 activity might therefore control neuronal morphogenesis in part by optimizing the levels of molecules that promote axon outgrowth.

    Funded by: NIAMS NIH HHS: AR38648, R01 AR038648, R37 AR038648; NINDS NIH HHS: NS33199, R37 NS033199

    Molecular and cellular biology 2005;25;13;5699-711

  • Munc18-1 stabilizes syntaxin 1, but is not essential for syntaxin 1 targeting and SNARE complex formation.

    Toonen RF, de Vries KJ, Zalm R, Südhof TC and Verhage M

    Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.

    Munc18-1, a member of the Sec1/Munc18 (SM) protein family, is essential for synaptic vesicle exocytosis. Munc18-1 binds tightly to the SNARE protein syntaxin 1, but the physiological significance and functional role of this interaction remain unclear. Here we show that syntaxin 1 levels are reduced by 70% in munc18-1 knockout mice. Pulse-chase analysis in transfected HEK293 cells revealed that Munc18-1 directly promotes the stability of syntaxin 1, consistent with a chaperone function. However, the residual syntaxin 1 in munc18-1 knockout mice is still correctly targeted to synapses and efficiently forms SDS-resistant SNARE complexes, demonstrating that Munc18-1 is not required for syntaxin 1 function as such. These data demonstrate that the Munc18-1 interaction with syntaxin 1 is physiologically important, but does not represent a classical chaperone-substrate relationship. Instead, the presence of SNARE complexes in the absence of membrane fusion in munc18-1 knockout mice indicates that Munc18-1 either controls the spatially correct assembly of core complexes for SNARE-dependent fusion, or acts as a direct component of the fusion machinery itself.

    Journal of neurochemistry 2005;93;6;1393-400

  • Baalc, a marker of mesoderm and muscle.

    Satoskar AA, Tanner SM, Weinstein M, Qualman SJ and de la Chapelle A

    Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, 420 West 12th Avenue, TMRF 646, Columbus, OH 43210, USA.

    Transcripts of the Brain and Acute Leukemia, Cytoplasmic (BAALC) gene are expressed in human neuroectodermal tissues and in CD34-positive bone marrow cells. High transcript levels occur in leukemic blasts from some patients with acute myeloid leukemia (AML), where high expression is an independent marker of poor prognosis. To gain insight into the hitherto unknown function of BAALC/Baalc, we studied its protein expression in embryonic and adult mouse tissue by immunohistochemical analysis. Baalc protein was mainly expressed in developing and mature muscle cells (cardiac, skeletal, and smooth) beginning on day E9 (heart). Signal was seen in the pre-muscle mesodermal cells of the dermatomyotome regions, and the derivatives of the lateral plate and intermediate mesoderm such as smooth muscle wall of the esophagus, stomach, the gut tube, bronchi, small blood vessels, and urinary bladder. This pattern continued through the late embryonic stages into adulthood. Baalc appeared to localize in the cytoplasm, adjacent to the cell membrane. This is distinctly observed in adult skeletal muscle cells. Baalc co-localized with known muscle-associated proteins but not with neural crest or neuronal markers. Scattered expression in adult bone marrow hematopoietic cells and weak expression in the brain neuropil also occurred. In conclusion, BAALC/Baalc is a marker of the mesodermal lineage, especially muscle.

    Funded by: NCI NIH HHS: CA09338 T32, CA098933, CA16058

    Gene expression patterns : GEP 2005;5;4;463-73

  • The RNA-binding protein HuD regulates neuronal cell identity and maturation.

    Akamatsu W, Fujihara H, Mitsuhashi T, Yano M, Shibata S, Hayakawa Y, Okano HJ, Sakakibara S, Takano H, Takano T, Takahashi T, Noda T and Okano H

    Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan.

    Neural Hu proteins (HuB/C/D) are RNA-binding proteins that have been shown to induce neuronal differentiation activity when overexpressed in immature neural progenitor cells or undifferentiated neuronal tumors. Newly generated HuD-deficient mice exhibited a transient impaired-cranial-nerve-development phenotype at an early embryonic stage. Adult HuD-deficient mice exhibited an abnormal hind-limb reflex and poor rotarod performance. Analysis of neurosphere formation revealed that the number and self-renewal capacity of the neural stem/progenitor cells were increased in HuD-deficient mice. HuD-deficient primary neurospheres also generated a smaller number of neurons. Cohort analysis of the cellular proliferative activity by using BrdUrd and iododeoxuridine labeling revealed that the number of differentiating quiescent cells in the embryonic cerebral wall was decreased. Long-term administration of BrdUrd revealed that the number of slowly dividing stem cells in the adult subventricular zone was increased in the HuD-deficient mice. Taken together, the results suggest that HuD is required at multiple points during neuronal development, including negative regulation of proliferative activity and neuronal cell-fate acquisition of neural stem/progenitor cells.

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;12;4625-30

  • Expression of the neuronal calcium sensor protein NCS-1 in the developing mouse olfactory pathway.

    Treloar HB, Uboha U, Jeromin A and Greer CA

    Department of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut 06520-8082, USA.

    Neuron specific calcium sensor 1 (NCS-1) is widely expressed in the developing and adult nervous system. Like calmodulin, NCS-1 is a member of a family of calcium binding proteins that contain EF-hand motifs, which bind calcium and induce conformational changes in the protein. Their binding varies with calcium concentration, allowing them to act as true calcium sensors rather than just calcium binding proteins. This family of proteins has been implicated in important synaptic events including neurotransmitter release and synapse formation. We examined the expression of NCS-1 in the developing and mature olfactory system to determine whether this molecule may be playing a role in establishing and/or maintaining olfactory circuitry. During development, expression of NCS-1 in the olfactory epithelium was localized in the dendritic knobs and axons of olfactory sensory neurons. Axonal expression was down-regulated after synapse formation. In the developing olfactory bulb, NCS-1 was expressed in the processes of mitral/tufted and granule cells. However, in the adult olfactory bulb, strongest expression was found in a subset of periglomerular cells (PGCs). This subset of PGCs did not express other known markers of PGCs including tyrosine hydroxylase, glutamic acid decarboxylase, calbindin, or calretinin, and only partially overlapped with the subpopulation of PGCs that express parvalbumin. Together, these data suggest multiple and overlapping roles of NCS-1 in the developing and mature olfactory system.

    Funded by: NIDCD NIH HHS: DC00210, DC03887, DC05706

    The Journal of comparative neurology 2005;482;2;201-16

  • Functional equivalence of Brn3 POU-domain transcription factors in mouse retinal neurogenesis.

    Pan L, Yang Z, Feng L and Gan L

    Center for Aging and Developmental Biology, University of Rochester, Rochester, NY 14642, USA.

    POU-domain transcription factors play essential roles in cell proliferation and differentiation. Previous studies have shown that targeted deletion of each of the three POU-domain Brn3 factors in mice leads to the developmental failure and apoptosis of a unique set of sensory neurons in retina, dorsal root ganglia, trigeminal ganglia and inner ear. The specific defects associated with the removal of each Brn3 gene closely reflect their characteristic spatiotemporal expression patterns. Nevertheless, it remains elusive whether Brn3 factors are functionally equivalent and act through a common molecular mechanism to regulate the development and survival of these sensory neurons. By knocking-in Brn3a (Brn3aki) into the Brn3b locus, we showed here that Brn3aki was expressed in a spatiotemporal manner identical to that of endogenous Brn3b. In addition, Brn3aki functionally restored the normal development and survival of retinal ganglion cells (RGCs) in the absence of Brn3b and fully reinstated the early developmental expression profiles of Brn3b downstream target genes in retina. These results indicate that Brn3 factors are functionally equal and that their unique roles in neurogenesis are determined by the distinctive Brn3 spatiotemporal expression patterns.

    Funded by: NEI NIH HHS: EY013426, EY015551, R01 EY013426, R01 EY013426-02, R01 EY015551, R01 EY015551-01

    Development (Cambridge, England) 2005;132;4;703-12

  • Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol.

    Vosseller K, Hansen KC, Chalkley RJ, Trinidad JC, Wells L, Hart GW and Burlingame AL

    Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA.

    While phosphorylation and O-GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post-translational modifications have been difficult to analyze in proteomic studies. We describe advances in beta-elimination / Michael addition-based approaches which allow for mass spectrometry-based identification and comparative quantification of O-phosphate or O-GlcNAc-modified peptides, as well as cysteine-containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope-coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O-phosphorylation and O-GlcNAc modification sites. Specificity of O-phosphate vs. O-GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O-GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post-translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large-scale quantitative analysis of both protein expression and serine / threonine post-translational modifications.

    Funded by: NCRR NIH HHS: RR-01614, RR-12961, RR-14606; NIGMS NIH HHS: P41 GM103481

    Proteomics 2005;5;2;388-98

  • Hippocampal-dependent memory is impaired in heterozygous GAP-43 knockout mice.

    Rekart JL, Meiri K and Routtenberg A

    Department of Psychology, Northwestern University, Evanston, Illinois 60208, USA.

    Cajal proposed that the rearrangement and growth of neurites and synaptic terminals is a substrate for the formation and storage of long-term memories. Proteins that regulate this learning-dependent growth are therefore likely to be "core determinants" (Sanes and Lichtman, Nat Neurosci 1999; 2:597-604) of such information storage processes. Although the growth-associated, protein kinase C (PKC) substrate GAP-43 has been oft-implicated in synaptic plasticity and memory, it has never been demonstrated that a reduction in the level of this protein has a deleterious effect on memory, because most homozygotes die perinatally. In this report, we observe significant memory impairments in heterozygous GAP-43 knockout mice with GAP-43 levels reduced by one-half. Impaired memory for a context was demonstrated in contextual fear conditioning. Importantly, no significant impairments in cued conditioning or on tests of nociceptive or auditory perception were observed in the heterozygous knockout, indicating that the observed impairments were unlikely related to performance or acquisition factors and are the result of reduced GAP-43 levels in the hippocampus. The present results, taken together with the prior demonstration of enhanced memory in transgenic mice overexpressing GAP-43, provide strong evidence for a pivotal role of hippocampal GAP-43 in the bidirectional regulation of mnemonic processing.

    Funded by: NIMH NIH HHS: MH 65436-01; NINDS NIH HHS: NS 33118; PHS HHS: TG 067564

    Hippocampus 2005;15;1;1-7

  • Identification of PSD-95 palmitoylating enzymes.

    Fukata M, Fukata Y, Adesnik H, Nicoll RA and Bredt DS

    Department of Physiology, University of California at San Francisco, San Francisco, California 94143, USA.

    Palmitoylation is a lipid modification that plays a critical role in protein trafficking and function throughout the nervous system. Palmitoylation of PSD-95 is essential for its regulation of AMPA receptors and synaptic plasticity. The enzymes that mediate palmitoyl acyl transfer to PSD-95 have not yet been identified; however, proteins containing a DHHC cysteine-rich domain mediate palmitoyl acyl transferase activity in yeast. Here, we isolated 23 mammalian DHHC proteins and found that a subset specifically palmitoylated PSD-95 in vitro and in vivo. These PSD-95 palmitoyl transferases (P-PATs) showed substrate specificity, as they did not all enhance palmitoylation of Lck, SNAP-25b, Galpha(s), or H-Ras in cultured cells. Inhibition of P-PAT activity in neurons reduced palmitoylation and synaptic clustering of PSD-95 and diminished AMPA receptor-mediated neurotransmission. This study suggests that P-PATs regulate synaptic function through PSD-95 palmitoylation.

    Neuron 2004;44;6;987-96

  • Myelination triggers local loss of axonal CNR/protocadherin alpha family protein expression.

    Morishita H, Kawaguchi M, Murata Y, Seiwa C, Hamada S, Asou H and Yagi T

    KOKORO Biology Group, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita 565-0871, Japan.

    The cadherin-related neuronal receptor (CNR)/protocadherin (Pcdh) alpha family is one of the diverse protocadherin families expressed in developing axons. We observed a strong axonal expression of these proteins at late embryonic and early postnatal stages corresponding to regions where fibers had not yet been myelinated. We therefore followed the postnatal localization of CNR/Pcdh alpha protein in major axonal tracts, such as the internal capsule, lateral olfactory tract, and optic nerve, and found that its axonal localization was dramatically lost in parallel with the increased expression of myelin markers. Moreover, the hypomyelinated optic nerve tracts of the myelin-deficient Shiverer mouse exhibited elevated levels of CNR/Pcdh alpha expression. These axonal expression patterns of CNR/Pcdh alpha in wild-type and Shiverer mice were similar to those of growth associated protein 43 (GAP-43) and L1, both of which are associated with axonal maturation. Thus, myelination may be a trigger for the local loss of axonal CNR/Pcdh alpha protein, and this process may be important in the maturation of neural circuits.

    The European journal of neuroscience 2004;20;11;2843-7

  • GFRalpha1 expression in cells lacking RET is dispensable for organogenesis and nerve regeneration.

    Enomoto H, Hughes I, Golden J, Baloh RH, Yonemura S, Heuckeroth RO, Johnson EM and Milbrandt J

    Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA. enomoto@cdb.riken.jp

    The GDNF family ligands signal through a receptor complex composed of a ligand binding subunit, GFRalpha, and a signaling subunit, the RET tyrosine kinase. GFRalphas are expressed not only in RET-expressing cells, but also in cells lacking RET. A body of evidence suggests that RET-independent GFRalphas are important for (1) modulation of RET signaling in a non-cell-autonomous fashion (trans-signaling) and (2) regulation of NCAM function. To address the physiological significance of these roles, we generated mice specifically lacking RET-independent GFRalpha1. These mice exhibited no deficits in regions where trans-signaling has been implicated in vitro, including enteric neurons, motor neurons, kidney, and regenerating nerves. Furthermore, no abnormalities were found in the olfactory bulb, which requires proper NCAM function for its formation and is putatively a site of GDNF-GFRalpha-NCAM signaling. Thus RET-independent GFRalpha1 is dispensable for organogenesis and nerve regeneration in vivo, indicating that trans-signaling and GFRalpha-dependent NCAM signaling play a minor role physiologically.

    Funded by: NIA NIH HHS: AG01373, AG13729; NIDDK NIH HHS: R01 DK57038, R01 DK64592

    Neuron 2004;44;4;623-36

  • Laminar organization of the developing lateral olfactory tract revealed by differential expression of cell recognition molecules.

    Inaki K, Nishimura S, Nakashiba T, Itohara S and Yoshihara Y

    Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, Saitama 351-0198, Japan.

    The projection neurons in the olfactory bulb (mitral and tufted cells) send axons through the lateral olfactory tract (LOT) onto several structures of the olfactory cortex. However, little is known of the molecular and cellular mechanisms underlying establishment of functional connectivity from the bulb to the cortex. Here, we investigated the developmental process of LOT formation by observing expression patterns of cell recognition molecules in embryonic mice. We immunohistochemically identified a dozen molecules expressed in the developing LOT and some of them were localized to subsets of mitral cell axons. Combinatorial immunostaining for these molecules revealed that the developing LOT consists of three laminas: superficial, middle, and deep. Detailed immunohistochemical, in situ hybridization, and 5-bromodeoxyuridine labeling analyses suggested that the laminar organization reflects: 1) the segregated pathways from the accessory and main olfactory bulbs, and 2) the different maturity of mitral cell axons. Mitral cell axons of the accessory olfactory bulb were localized to the deep lamina, segregated from those of the main olfactory bulb. In the main olfactory pathway, axons of mature mitral cells, whose somata is located in the apical sublayer of the mitral cell layer, were localized to the middle lamina within LOT, while those of immature mitral cells that located in the basal sublayer were complementarily localized to the superficial lamina. These results suggest that newly generated immature axons are added to the most superficial lamina of LOT successively, leading to the formation of piled laminas with different maturational stages of the mitral cell axons.

    The Journal of comparative neurology 2004;479;3;243-56

  • The Lim homeobox gene Lhx2 is required for olfactory sensory neuron identity.

    Kolterud A, Alenius M, Carlsson L and Bohm S

    Department of Molecular Biology, Umeå University, Umeå, SE901 87, Sweden.

    Progenitor cells in the mouse olfactory epithelium generate over a thousand subpopulations of neurons, each expressing a unique odorant receptor (OR) gene. This event is under the control of spatial cues, since neurons in different epithelial regions are restricted to express region-specific subsets of OR genes. We show that progenitors and neurons express the LIM-homeobox gene Lhx2 and that neurons in Lhx2-null mutant embryos do not diversify into subpopulations expressing different OR genes and other region-restricted genes such as Nqo1 and Ncam2. Lhx2-/- embryos have, however, a normal distribution of Mash1-positive and neurogenin 1-positive neuronal progenitors that leave the cell cycle, acquire pan-neuronal traits and form axon bundles. Increased cell death in combination with increased expression of the early differentiation marker Neurod1, as well as reduced expression of late differentiation markers (Galphaolf and Omp), suggests that neuronal differentiation in the absence of Lhx2 is primarily inhibited at, or immediate prior to, onset of OR expression. Aberrant regional expression of early and late differentiation markers, taken together with unaltered region-restricted expression of the Msx1 homeobox gene in the progenitor cell layer of Lhx2-/- embryos, shows that Lhx2 function is not required for all aspects of regional specification of progenitors and neurons. Thus, these results indicate that a cell-autonomous function of Lhx2 is required for differentiation of progenitors into a heterogeneous population of individually and regionally specified mature olfactory sensory neurons.

    Development (Cambridge, England) 2004;131;21;5319-26

  • Phosphoproteomic analysis of the developing mouse brain.

    Ballif BA, Villén J, Beausoleil SA, Schwartz D and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.

    Funded by: NHGRI NIH HHS: HG00041

    Molecular & cellular proteomics : MCP 2004;3;11;1093-101

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Transdifferentiation of the retina into pigmented cells in ocular retardation mice defines a new function of the homeodomain gene Chx10.

    Rowan S, Chen CM, Young TL, Fisher DE and Cepko CL

    Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

    The homeodomain transcription factor Chx10 is one of the earliest markers of the developing retina. It is required for retinal progenitor cell proliferation as well as formation of bipolar cells, a type of retinal interneuron. or(J) (ocular retardation) mice, which are Chx10 null mutants, are microphthalmic and show expanded and abnormal peripheral structures, including the ciliary body. We show here, in a mixed genetic background, the progressive appearance of pigmented cells in the neural retina, concomitant with loss of expression of retinal markers. Fate mapping analysis using a multifunctional Chx10 BAC reporter mouse revealed this process to be direct transdifferentiation of retinal cells into pigmented cells. Microarray and in situ hybridization analyses revealed a complex program underlying the transdifferentiation. This program involved the expansion of expression of genes normally found only in the periphery into central regions of the eye. These genes included a transcription factor controlling pigmentation, Mitf, and the related factor Tfec (Tcfec -- Mouse Genome Informatics), which can activate a melanogenic gene expression program. Misexpression of Chx10 in the developing retinal pigmented epithelium (RPE) caused downregulation of Mitf, Tfec, and associated pigment markers, leading to a nonpigmented RPE. These data link Chx10 and Mitf to maintenance of the neural retina and RPE fates respectively. Further, they suggest a new role for Chx10 in maintenance of compartment boundaries in the peripheral retina.

    Development (Cambridge, England) 2004;131;20;5139-52

  • Expression levels of cytoskeletal proteins indicate pathological aging of S100B transgenic mice: an immunohistochemical study of MAP-2, drebrin and GAP-43.

    Shapiro LA and Whitaker-Azmitia PM

    Program in Biopsychology, Department of Psychology, State University of New York, Stony Brook, NY 11794-2500, USA.

    S100B is a calcium-binding protein found within astroglial cells. When released, S100B has extracellular neurotrophic effects involving the neuronal cytoskeleton. The gene for S100B is located on chromosome 21 and levels of the protein are elevated in Down Syndrome (DS) and Alzheimer's Disease (AD). Thus, overexpression of S100B may be related to the cytoskeletal abnormalities seen in these disorders. Transgenic mice overexpressing human S100B were examined for cytoskeletal changes as young (70 days) and aged (200 days) adults, using immunochemical staining of the dendritic associated protein, MAP-2, the growth-associated protein-43 (GAP-43) and the dendritic spine marker, drebrin. As young adults, the S100B transgenic mice exhibited significantly greater MAP-2-immunoreactivity in the hippocampus, however as older adults, the animals exhibited less staining. In both the CD1 control animals and the S100B animals, the immunoreactivity of drebrin increased with age, however there were no significant between group differences. Finally, the older S100B animals showed more GAP-43 staining than the control animals, suggesting that synaptic remodeling could take place, possibly in response to the loss of MAP-2-ir dendrites. Overall, the data suggest that S100B overexpression leads to changes in cytoskeletal markers. The longitudinal effects of S100B overexpression are discussed with relevance to aging and pathology.

    Funded by: NINDS NIH HHS: R01 NS 042555

    Brain research 2004;1019;1-2;39-46

  • Co-induction of growth-associated protein GAP-43 and neuronal nitric oxide synthase in the cochlear nucleus following cochleotomy.

    Chen TJ, Huang CW, Wang DC and Chen SS

    Department of Physiology, Kaohsiung Medical University, 807, Kaohsiung, Taiwan.

    In adult animals, cochlear lesioning leads to a reactive synaptogenesis with a reemergence of growth-associated protein, GAP-43, in the auditory brainstem nuclei. In addition, nitric oxide (NO) is also implicated in synaptogenesis. Three isoforms of nitric oxide synthase (NOS) responsible for generating NO have been identified and, in neurons, the predominant isoform is neuronal NOS (nNOS). Studies in visual or olfactory systems have found that the NOS expression often correlates with periods of axonal outgrowth and synapse formation; whether NO plays a similar role in the auditory brainstem needs to be examined. In the present study, a unilateral cochleotomy was performed in adult mice to examine the relationship between the reemergence of GAP-43 and the expression pattern of nNOS. Following surgery, GAP-43 re-emerged in the ipsilateral anterior ventral cochlear nucleus (AVCN) and the immunoreactivity reached a climax around postoperative day (POD) 8; the same expression pattern as that reported in the previous literature is the indicator of synaptogenesis. As for the nNOS immunoreactivity, a dramatic redistribution from a mostly cytoplasmal to a predominantly membranous localization in the ipsilateral AVCN was found especially at POD 4. A similar redistribution pattern in the ipsilateral AVCN for the N-methyl-D-aspartate (NMDA) receptor was also observed at POD 4, corresponding to the fact that the activation of nNOS is coupled to calcium influx via the NMDA-receptor. Furthermore, the expression of cyclic guanosine monophosphate (cGMP) is an indicator for activity of soluble guanylyl cyclase (sGC), the substrate of NO, which reveals the target area of NO. Therefore, cGMP immunoreactivity was also examined and an obvious increase of cytoplasmal cGMP expression was observed around POD 4. Accordingly, it is suggested that nNOS activity correlates closely with the reactive synaptogenesis following a cochleotomy. Further evidence is shown by the results of fluorescent double staining; nNOS-positive cells were surrounded by GAP-43 labeled regions that appeared to be presynaptic boutons, and the vast majority of nNOS-positive cells also expressed cGMP. The former result indicates that, after surgery, there should be new terminal endings projecting onto the nNOS-positive cells in the AVCN. Furthermore, the latter result suggests a possible role of an autocrine mediator for nNOS in the AVCN.

    Experimental brain research 2004;158;2;151-62

  • Expression of olfactory receptors in the cribriform mesenchyme during prenatal development.

    Schwarzenbacher K, Fleischer J, Breer H and Conzelmann S

    Institute of Physiology, University of Hohenheim, Garbenstr. 30, 70599 Stuttgart, Germany.

    Olfactory receptors (ORs) are expressed in sensory neurons of the nasal epithelium, where they are supposed to be involved in the recognition of suitable odorous compounds and in the guidance of outgrowing axons towards the appropriate glomeruli in the olfactory bulb. During development, some olfactory receptor subtypes have also been found in non-sensory tissues, including the cribriform mesenchyme between the prospective olfactory epithelium and the developing telencephalon, but it is elusive if this is a typical phenomenon for ORs. Monitoring the onset and time course of expression for several receptor subtypes revealed that 'extraepithelial' expression of ORs occurs very early and transiently, in particular between embryonic stages E10.25 and E14.0. In later stages, a progressive loss of receptor expressing cells was observed. Molecular phenotyping demonstrated that the receptor expressing cells in the cribriform mesenchyme co-express key elements, including Galpha(olf), ACIII and OMP, characteristic for olfactory neurons in the nasal epithelium. Studies on transgenic OMP/GFP-mice showed that 'extraepithelial' OMP/GFP-positive cells are located in close vicinity to axon bundles projecting from the nasal epithelium to the presumptive olfactory bulb. Moreover, these cells are primarily located where axons fasciculate and change direction towards the anterior part of the forebrain.

    Gene expression patterns : GEP 2004;4;5;543-52

  • Failure to express GAP-43 leads to disruption of a multipotent precursor and inhibits astrocyte differentiation.

    Shen Y, Mani S and Meiri KF

    Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, MA 02111, USA.

    The nervous system-specific protein GAP-43 is significantly upregulated in neurons and glia that are differentiating. In P19 EC cells that do not express GAP-43, neurogenesis is inhibited; many immature neurons apoptose and the survivors do not mature morphologically. Here we show that the initial defect is in an early precursor with characteristics of a neural stem cell, which failed to respond normally to retinoic acid (RA). As a consequence, its progeny had altered cell fates: In addition to the neuronal defects previously reported, RC1-labeled radial glia failed to exit the cell cycle, accumulated, and failed to acquire GFAP immunoreactivity. However, leukemia inhibitory factor (LIF) could stimulate GFAP expression suggesting that astrocytes not derived from radial glia are less affected by absence of GAP-43. Differentiation of radial glia-derived astrocytes was also inhibited in glial cultures from GAP-43 (-/-) cerebellum, and in GAP-43 (-/-) telencephalon in vivo, differentiation of astrocytes derived from both radial and nonradial glia lineages were both affected: In the glial wedge, GFAP-labeled radial glia-derived astrocytes were reduced consistent with the interpretation that they may be unable to deflect GAP-43 (-/-) commissural axons toward the midline. At the midline, both radial and nonradial glia-derived astrocytes were also decreased although it fused normally. The results demonstrate that GAP-43 expressed in multipotent precursors is required for appropriate cell fate commitment, and that its absence affects astrocyte as well as neuronal differentiation.

    Funded by: NINDS NIH HHS: NS33118

    Molecular and cellular neurosciences 2004;26;3;390-405

  • Reciprocal changes of CD44 and GAP-43 expression in the dentate gyrus inner molecular layer after status epilepticus in mice.

    Borges K, McDermott DL and Dingledine R

    Department of Pharmacology, Emory University School of Medicine, Atlanta GA 30322, USA. kborges@pharm.emory.edu

    Mossy fiber sprouting (MFS), a common feature of human temporal lobe epilepsy and many epilepsy animal models, contributes to hippocampal hyperexcitability. The molecular events responsible for MFS are not well understood, although the growth-associated protein GAP-43 has been implicated in rats. Here, we focus on the hyaluronan receptor CD44, which is involved in routing of retinal axons during development and is upregulated after injury in many tissues including brain. After pilocarpine-induced status epilepticus (SE) in mice most hilar neurons died and neuropeptide Y (NPY) immunoreactivity appeared in the dentate inner molecular layer (IML) after 10-31 days indicative of MFS. Strong CD44 immunoreactivity appeared in the IML 3 days after pilocarpine, then declined over the next 4 weeks. Conversely, GAP-43 immunoreactivity was decreased in the IML at 3-10 days after pilocarpine-induced SE. After SE induced by repeated kainate injections, mice did not show any hilar cell loss or changes in CD44 or GAP-43 expression in the IML, and MFS was absent at 20-35 days. Thus, after SE in mice, early loss of GAP-43 and strong CD44 induction in the IML correlated with hilar cell loss and subsequent MFS. CD44 is one of the earliest proteins upregulated in the IML and coincides with early sprouting of mossy fibers, although its function is still unknown. We hypothesize that CD44 is involved in the response to axon terminal degeneration and/or neuronal reorganization preceding MFS.

    Funded by: NINDS NIH HHS: NS17771

    Experimental neurology 2004;188;1;1-10

  • Zinc finger gene fez-like functions in the formation of subplate neurons and thalamocortical axons.

    Hirata T, Suda Y, Nakao K, Narimatsu M, Hirano T and Hibi M

    Department of Molecular Oncology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

    fez-like (fezl) is a forebrain-expressed zinc finger gene required for the formation of the hypothalamic dopaminergic and serotonergic (monoaminergic) neurons in zebrafish. To reveal its function in mammals, we analyzed the expression of the mouse orthologue of fezl and generated fezl-deficient mice by homologous recombination. Mouse fezl was expressed specifically in the forebrain from embryonic day 8.5. At mid-gestation, fezl expression was detected in subdomains of the forebrain, including the dorsal telencephalon and ventral diencephalon. Unlike the zebrafish fezl mutant too few, the fezl-deficient mice displayed normal development of hypothalamic monoaminergic neurons, but showed abnormal "hyperactive" behavior. In fezl(-/-) mice, the thalamocortical axons (TCA) were reduced in number and aberrantly projected to the cortex. These mutants had a reduced number of subplate neurons, which are involved in guiding the TCA from the dorsal thalamus, although the subplate neurons were born normally. These results suggest that fezl is required for differentiation or survival of the subplate neurons, and reduction of the subplate neurons in fezl-deficient mice leads to abnormal development of the TCA, providing a possible link between the transcriptional regulation of forebrain development and hyperactive behavior.

    Developmental dynamics : an official publication of the American Association of Anatomists 2004;230;3;546-56

  • The LIM-homeodomain protein Lhx2 is required for complete development of mouse olfactory sensory neurons.

    Hirota J and Mombaerts P

    The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

    In mice, approximately 1,000 odorant receptor (OR) genes are expressed in olfactory sensory neurons (OSNs). Homeodomain sites can be recognized in the promoter and upstream regions of several OR genes. Here, using the yeast one-hybrid system and electrophoretic mobility shift assay, we report that Lhx2, a LIM-homeodomain protein, binds to the homeodomain site in the mouse M71 OR promoter region. In Lhx2-deficient mice, the morphology of the olfactory epithelium is grossly normal. However, expression of OMP is abolished and that of GAP43 is severely reduced, indicating that no mature and few immature OSNs are produced. M71 and other OR genes also are not expressed. OSN development appears to be arrested between the terminal differentiation into neurons and the transition to immature neurons. Thus, Lhx2 is required for complete development of OSNs in mice.

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;23;8751-5

  • Development of the mouse hypothalamo-neurohypophysial system in the munc18-1 null mutant that lacks regulated secretion.

    Korteweg N, Maia AS, Verhage M and Burbach JP

    Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands.

    The hypothalamo-neurohypophysial system (HNS) is composed of hypothalamic magnocellular neurons and neural lobe pituicytes that accommodate the nerve terminals. Here we have investigated if the communication of the peptidergic neurons of the HNS with neighbouring cells plays a role in the development and assembly of the HNS. We employed munc18-1-deficient mice, which completely lack neurotransmitter secretion. Morphological and immunohistological analysis of the HNS in these mutant embryos during brain development showed that this peptidergic system was formed normally during early embryogenesis. However, the development arrested at embryonal day 14.5, the stage when terminal differentiation has to take place. The peptidergic neurons targeted axons in the correct direction, but few arrived at their final location and the neurons were not maintained in later stages. The pituicytes in the neural lobe of the pituitary were generated, but failed to organize normally. Our results indicate that peptide gene expression, axon outgrowth and migration are intrinsic developmental events in these peptidergic neurons, that are initiated in the munc18-1 null mutant. The further expansion and the integration of outgrowing axon terminals with neural lobe pituicytes requires munc18-1-dependent processes, probably exocytosis, at multiple levels. Firstly, to maintain and propagate neuronal outgrowth and guidance, and secondly, to control the cellular organization of the pituicytes. Thus, the communication between the outgrowing neurons and the pituicytes could serve to integrate these two cell types to constitute a functional peptidergic system.

    The European journal of neuroscience 2004;19;11;2944-52

  • Msx1-deficient mice fail to form prosomere 1 derivatives, subcommissural organ, and posterior commissure and develop hydrocephalus.

    Fernández-Llebrez P, Grondona JM, Pérez J, López-Aranda MF, Estivill-Torrús G, Llebrez-Zayas PF, Soriano E, Ramos C, Lallemand Y, Bach A and Robert B

    Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain. llebrez@uma.es

    Msx1 is a regulatory gene involved in epithelio-mesenchymal interactions in limb formation and organogenesis. In the embryonic CNS, the Msx1 gene is expressed along the dorsal midline. Msx1 mutant mice have been obtained by insertion of the nlacZ gene in the Msx1 homeodomain. The most important features of homozygous mutants that we observed were the absence or malformation of the posterior commissure (PC) and of the subcommissural organ (SCO), the collapse of the cerebral aqueduct, and the development of hydrocephalus. Heterozygous mutants developed abnormal PC and reduced SCO, as revealed by specific antibodies against SCO secretory glycoproteins. About one third of the heterozygous mutants also showed hydrocephalus. Other defects displayed by homozygous mutants were ependymal denudation, subventricular cavitations and edema, and underdevelopment of the pineal gland and subfornical organ. Some homozygous mutants developed both SCO and PC, probably as a consequence of genetic redundancy with Msx2. However, these mutants did not show SCO-immunoreactive glycoproteins and displayed obstructive hydrocephalus. This suggests that Msx1 is necessary for the synthesis of SCO glycoproteins, which would then be required for the maintenance of an open aqueduct.

    Journal of neuropathology and experimental neurology 2004;63;6;574-86

  • Loss of Gli3 and Shh function disrupts olfactory axon trajectories.

    Balmer CW and LaMantia AS

    Department of Cell & Molecular Physiology, Curriculum in Neurobiology and University of North Carolina Neuroscience Center, University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, North Carolina 27599, USA.

    The transcriptional regulator Gli3 and the secreted signal Shh influence induction, patterning, and differentiation at several sites of mesenchymal/epithelial (M/E) interaction including the limbs, heart, face, and forebrain. We asked whether loss of function of these two genes has specific consequences for early differentiation of the primary olfactory pathway-which comprises both craniofacial and forebrain structures and depends on M/E induction during initial stages of development. Loss of Gli3 or Shh function does not compromise several aspects of olfactory receptor neuron (ORN) and olfactory ensheathing cell maturation; however, directed outgrowth of ORN axons and their initial targeting to the telencephalon is altered. In Gli3 mutant extra toes-Jackson (Xt(J)Xt(J)) embryos, ORN axons defasciculate and project aberrantly near the forebrain. They rarely enter the central nervous system, and their association with mesenchymal laminin is disrupted. In Shh-/-embryos, ORN axons exit a single olfactory epithelium (OE) that develops centrally within an altered mesenchymal environment in a dysmorphic proboscis. These axons project as a single nerve toward the mutant forebrain; however, their trajectory varies according to the position of the proboscis relative to the forebrain. These alterations in axon outgrowth probably reflect compromised inductive interactions in the olfactory primordia because neither Gli3 nor Shh are expressed in olfactory neurons. Thus, two genes that influence induction and subsequent differentiation of craniofacial structures and the forebrain have distinct consequences for ORN axon growth during the initial genesis of the olfactory pathway.

    Funded by: NICHD NIH HHS: HD29178

    The Journal of comparative neurology 2004;472;3;292-307

  • Notch expression in developing olfactory neuroepithelium.

    Doi K, Ishida H and Nibu K

    Department of Otolaryngology-Head and Neck Surgery, Kobe University Graduate School of Medicine, Kusunoki-cho 7-5-1, Chuo-ku, Kobe 650-0017, Japan.

    Notch genes encode receptors for signaling pathways that regulate neurogenesis in various tissues. To better understand the roles of Notch genes in olfactory neurogenesis, we studied the expression of Notch family in developing mouse olfactory epithelium. In the earliest stage of olfactory development, Notch1 was observed in the mesenchyme lateral to the olfactory placode. During the developmental stage, Notch1 was mainly observed around the basal membrane, while Notch3 was observed in the lower compartment of the olfactory neuroepithelium. Notch2 was not detected during the entire observation period. As the olfactory neuroepithelium grew mature, both Notch1 and Notch3 gradually disappeared. These results suggest distinct roles of Notch1 and Notch3 in the neurogenesis of the peripheral olfactory system.

    Neuroreport 2004;15;6;945-7

  • Molecular determinants of synapsin targeting to presynaptic terminals.

    Gitler D, Xu Y, Kao HT, Lin D, Lim S, Feng J, Greengard P and Augustine GJ

    Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Although synapsins are abundant synaptic vesicle proteins that are widely used as markers of presynaptic terminals, the mechanisms that target synapsins to presynaptic terminals have not been elucidated. We have addressed this question by imaging the targeting of green fluorescent protein-tagged synapsins in cultured hippocampal neurons. Whereas all synapsin isoforms targeted robustly to presynaptic terminals in wild-type neurons, synapsin Ib scarcely targeted in neurons in which all synapsins were knocked-out. Coexpression of other synapsin isoforms significantly strengthened the targeting of synapsin Ib in knock-out neurons, indicating that heterodimerization is required for synapsin Ib to target. Truncation mutagenesis revealed that synapsin Ia targets via distributed binding sites that include domains B, C, and E. Although domain A was not necessary for targeting, its presence enhanced targeting. Domain D inhibited targeting, but this inhibition was overcome by domain E. Thus, multiple intermolecular and intramolecular interactions are required for synapsins to target to presynaptic terminals.

    Funded by: NIMH NIH HHS: MH 39327, MH 67044

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;14;3711-20

  • Discrete gene sets depend on POU domain transcription factor Brn3b/Brn-3.2/POU4f2 for their expression in the mouse embryonic retina.

    Mu X, Beremand PD, Zhao S, Pershad R, Sun H, Scarpa A, Liang S, Thomas TL and Klein WH

    Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

    Brn3b/Brn-3.2/POU4f2 is a POU domain transcription factor that is essential for retinal ganglion cell (RGC) differentiation, axonal outgrowth and survival. Our goal was to establish a link between Brn3b and the downstream events leading to RGC differentiation. We sought to determine both the number and types of genes that depend on Brn3b for their expression. RNA probes from wild-type and Brn3b(-/-) E14.5, E16.5 and E18.5 mouse retinas were hybridized to a microarray containing 18,816 retina-expressed cDNAs. At E14.5, we identified 87 genes whose expression was significantly altered in the absence of Brn3b and verified the results by real-time PCR and in situ hybridization. These genes fell into discrete sets that encoded transcription factors, proteins associated with neuron integrity and function, and secreted signaling molecules. We found that Brn3b influenced gene expression in non RGCs of the retina by controlling the expression of secreted signaling molecules such as sonic hedgehog and myostatin/Gdf8. At later developmental stages, additional alterations in gene expression were secondary consequences of aberrant RGC differentiation caused by the absence of Brn3b. Our results demonstrate that a small but crucial fraction of the RGC transcriptome is dependent on Brn3b. The Brn3b-dependent gene sets therefore provide a unique molecular signature for the developing retina.

    Funded by: NCI NIH HHS: CA16672; NEI NIH HHS: EY11930, EY13523

    Development (Cambridge, England) 2004;131;6;1197-210

  • Endothelin-1 regulates cardiac sympathetic innervation in the rodent heart by controlling nerve growth factor expression.

    Ieda M, Fukuda K, Hisaka Y, Kimura K, Kawaguchi H, Fujita J, Shimoda K, Takeshita E, Okano H, Kurihara Y, Kurihara H, Ishida J, Fukamizu A, Federoff HJ and Ogawa S

    Cardiopulmonary Division, Department of Internal Medicine, Keio University School of Medicine, Tookyo, Japan.

    The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-1 specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-1-induced NGF augmentation was mediated by the endothelin-A receptor, Gibetagamma, PKC, the Src family, EGFR, extracellular signal-regulated kinase, p38MAPK, activator protein-1, and the CCAAT/enhancer-binding protein delta element. Either conditioned medium or coculture with endothelin-1-stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-1-deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-1-deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late embryonic stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-1-deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-1 regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.

    The Journal of clinical investigation 2004;113;6;876-84

  • GAP-43 overexpression in adult mouse Purkinje cells overrides myelin-derived inhibition of neurite growth.

    Gianola S and Rossi F

    Rita Levi Montalcini Centre for Brain Repair, Department of Neuroscience, University of Turin, Corso Raffaello 30, I-10125 Turin, Italy.

    Up-regulation of growth-associated proteins in adult neurons promotes axon regeneration and neuritic elongation onto nonpermissive substrates. To investigate the interaction between these molecules and myelin-related inhibitory factors, we examined transgenic mice in which overexpression of the growth-associated protein GAP-43 is driven by the Purkinje cell-specific promoter L7. Contrary to their wild-type counterparts, which have extremely poor regenerative capabilities, axotomized transgenic Purkinje cells exhibit profuse sprouting along the intracortical neurite and at the severed stump [Buffo et al. (1997) J. Neurosci., 17, 8778-8791]. Here, we investigated the relationship between such sprouting axons and oligodendroglia to ask whether GAP-43 overexpression enables Purkinje neurites to overcome myelin-derived inhibition. Intact transgenic Purkinje axons display normal morphology and myelination. Following injury, however, many GAP-43-overexpressing neurite stumps are devoid of myelin cover and sprout into white matter regions containing densely packed myelin and Nogo-A- or MAG-immunopositive oligodendrocytes. The intracortical segments of these neurites show focal accumulations of GAP-43, which are associated with disrupted or retracted myelin sheaths. Numerous sprouts originate from such demyelinated segments and spread into the granular layer. Some myelin loss, though not axon sprouting, is also evident in wild-type mice, but this phenomenon is definitely more rapid and extensive in transgenic cerebella. Thus, GAP-43-overexpressing Purkinje axons are endowed with enhanced capabilities for growing into nonpermissive territories and show a pronounced tendency to lose myelin. Our observations suggest that accumulation of GAP-43 along precise axon segments disrupts the normal axon-glia interaction and enhances the retraction of oligodendrocytic processes to facilitate the outgrowth of neuritic sprouts.

    The European journal of neuroscience 2004;19;4;819-30

  • Repulsive guidance molecule (RGM) gene function is required for neural tube closure but not retinal topography in the mouse visual system.

    Niederkofler V, Salie R, Sigrist M and Arber S

    Biozentrum, Department of Cell Biology, University of Basel, 4056 Basel, Switzerland

    The establishment of topographic projections in the developing visual system depends on the spatially and temporally controlled expression of axon guidance molecules. In the developing chick tectum, the graded expression of the repulsive guidance molecule (RGM) has been proposed to be involved in controlling the topography of the retinal ganglion cell (RGC) axon termination zones along the anteroposterior axis of the tectum. We now show that there are three mouse proteins homologous to chick RGM displaying similar proteolytic processing but exhibiting differential cell-surface targeting by glycosyl phosphatidylinositol anchor addition. Two members of this gene family (mRGMa and mRGMb) are expressed in complementary patterns in the nervous system, and mRGMa is expressed prominently in the superior colliculus at the time of anteroposterior targeting of RGC axons. The third member of the family (mRGMc) is expressed almost exclusively in skeletal muscles. Functional studies in the mouse reveal a role for mRGMa in controlling cephalic neural tube closure, thus defining an unexpected role for mRGMa in early embryonic development. In contrast, mRGMa mutant mice did not exhibit defects in anteroposterior targeting of RGC axons to their stereotypic termination zones in the superior colliculus.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;4;808-18

  • Behavioral characterization in a comprehensive mouse test battery reveals motor and sensory impairments in growth-associated protein-43 null mutant mice.

    Metz GA and Schwab ME

    Canadian Center for Behavioural Neuroscience, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta, T1K 3M4 Canada. gerlinde.metz@uleth.ca

    The growth-associated protein (GAP)-43 is a major neuronal protein associated with axonal growth, neuronal plasticity and learning. The observation that only 5-10% of mice with a full GAP-43 gene deletion survive weaning suggests that basic neural functions are disturbed. Here we used a comprehensive test battery to characterise and quantify the motor and sensory function of surviving adult homozygous GAP-43 (-/-) mice as compared with GAP-43 (+/-) and wild-type animals. The test battery was comprised of motor, sensory, and reflex tests producing 25 measures of locomotion, as well as epicritic, auditory, olfactory and visual function. The analysis revealed significant impairments in muscle strength, limb coordination and balance in GAP-43 (-/-) mice. Furthermore, GAP-43 (-/-) animals were hyperactive and showed reduced anxiety as measured by open field and light dark tests. In sensory tests, GAP-43 (-/-) mice were tested for impaired tactile and labyrinthine function. Abnormal reflexes were found in the contact and vibrissa placing responses, and in the crossed extensor reflex. GAP-43 (+/-) animals showed only moderate abnormalities as compared with wild-type animals. We conclude that GAP-43 is necessary for the development and function of a variety of neuronal systems. The results also show that the comprehensive test battery used in the present study represents a sensitive approach to assess the functional integrity of ascending and descending pathways in genetically manipulated mice.

    Neuroscience 2004;129;3;563-74

  • A comprehensive analysis of 22q11 gene expression in the developing and adult brain.

    Maynard TM, Haskell GT, Peters AZ, Sikich L, Lieberman JA and LaMantia AS

    Neurodevelopmental Disorders Research Center, Silvio O. Conte Center for the Neuroscience of Mental Disorders, Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.

    Deletions at 22q11.2 are linked to DiGeorge or velocardiofacial syndrome (VCFS), whose hallmarks include heart, limb, and craniofacial anomalies, as well as learning disabilities and increased incidence of schizophrenia. To assess the potential contribution of 22q11 genes to cognitive and psychiatric phenotypes, we determined the CNS expression of 32 mouse orthologs of 22q11 genes, primarily in the 1.5-Mb minimal critical region consistently deleted in VCFS. None are uniquely expressed in the developing or adult mouse brain. Instead, 27 are localized in the embryonic forebrain as well as aortic arches, branchial arches, and limb buds. Each continues to be expressed at apparently constant levels in the fetal, postnatal, and adult brain, except for Tbx1, ProDH2, and T10, which increase in adolescence and decline in maturity. At least six 22q11 proteins are seen primarily in subsets of neurons, including some in forebrain regions thought to be altered in schizophrenia. Thus, 22q11 deletion may disrupt expression of multiple genes during development and maturation of neurons and circuits compromised by cognitive and psychiatric disorders associated with VCFS.

    Funded by: NICHD NIH HHS: HD-29178, HD-40127, R01 HD029178, R29 HD029178, T32 HD040127; NIMH NIH HHS: P50 MH033127

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14433-8

  • Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention.

    Zambrowicz BP, Abuin A, Ramirez-Solis R, Richter LJ, Piggott J, BeltrandelRio H, Buxton EC, Edwards J, Finch RA, Friddle CJ, Gupta A, Hansen G, Hu Y, Huang W, Jaing C, Key BW, Kipp P, Kohlhauff B, Ma ZQ, Markesich D, Payne R, Potter DG, Qian N, Shaw J, Schrick J, Shi ZZ, Sparks MJ, Van Sligtenhorst I, Vogel P, Walke W, Xu N, Zhu Q, Person C and Sands AT

    Lexicon Genetics, 8800 Technology Forest Place, The Woodlands, TX 77381, USA. brian@lexgen.com

    The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14109-14

  • Abnormal thalamocortical pathfinding and terminal arbors lead to enlarged barrels in neonatal GAP-43 heterozygous mice.

    McIlvain VA, Robertson DR, Maimone MM and McCasland JS

    Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.

    GAP-43 has been implicated in axonal pathfinding and sprouting, synaptic plasticity, and neurotransmitter release. However, its effect on cortical development in vivo is poorly understood. We have previously shown that GAP-43 knockout (-/-) mice fail to develop whisker-related barrels or an ordered whisker map in the cortex. Here we used cytochrome oxidase (CO) histochemistry to demonstrate that GAP-43 heterozygous (+/-) mice develop larger than normal barrels at postnatal day 7 (P7), despite normal body and brain weight. Using serotonin transporter (5HT-T) histochemistry to label thalamocortical afferents (TCAs), we found no obvious abnormalities in other somatosensory areas or primary visual cortex of GAP-43 (+/-) mice. However, TCA projections to (+/-) primary auditory cortex were not as clearly defined. To clarify the mechanism underlying the large-barrel phenotype, we used lipophilic (DiI) axon labeling. We found evidence for multiple pathfinding abnormalities among GAP-43 (+/-) TCAs. These axons show increased fasciculation within the internal capsule, as well as abnormal turning and branching in the subcortical white matter. These pathfinding errors most likely reflect failures of signal recognition and/or transduction by ingrowing TCAs. In addition, many DiI-labeled (+/-) TCAs exhibit widespread, sparsely branched terminal arbors in layer IV, reflecting the large-barrel phenotype. They also resemble those found in rat barrel cortex deprived of whisker inputs from birth, suggesting a failure of activity-dependent synaptogenesis and/or synaptic stabilization in (+/-) cortex. Our findings suggest that reduced GAP-43 expression can alter the fine-tuning of a cortical map through a combination of pathfinding and synaptic plasticity mechanisms.

    Funded by: NINDS NIH HHS: NS31829, NS40779

    The Journal of comparative neurology 2003;462;2;252-64

  • Back signaling by the Nrg-1 intracellular domain.

    Bao J, Wolpowitz D, Role LW and Talmage DA

    Institute of Human Nutrition, 701 West 168th St., 5-503 New York, NY 10032, USA. dat1@columbia.edu

    Transmembrane isoforms of neuregulin-1 (Nrg-1), ligands for erbB receptors, include an extracellular domain with an EGF-like sequence and a highly conserved intracellular domain (ICD) of unknown function. In this paper, we demonstrate that transmembrane isoforms of Nrg-1 are bidirectional signaling molecules in neurons. The stimuli for Nrg-1 back signaling include binding of erbB receptor dimers to the extracellular domain of Nrg-1 and neuronal depolarization. These stimuli elicit proteolytic release and translocation of the ICD of Nrg-1 to the nucleus. Once in the nucleus, the Nrg-1 ICD represses expression of several regulators of apoptosis, resulting in decreased neuronal cell death in vitro. Thus, regulated proteolytic processing of Nrg-1 results in retrograde signaling that appears to mediate contact and activity-dependent survival of Nrg-1-expressing neurons.

    Funded by: NCI NIH HHS: CA79737, R01 CA079737; NIA NIH HHS: AG01016, K01 AG001016; NIDDK NIH HHS: DK07715, T32 DK007715; NINDS NIH HHS: NS29071, R01 NS029071, R01 NS029071-14, R01 NS029071-15, R01 NS029071-16, R01 NS029071-17

    The Journal of cell biology 2003;161;6;1133-41

  • The Dlx5 homeodomain gene is essential for olfactory development and connectivity in the mouse.

    Levi G, Puche AC, Mantero S, Barbieri O, Trombino S, Paleari L, Egeo A and Merlo GR

    Laboratoire de Physiologie Général et Comparée, CNRS, UMR 8572, Museum National d'Histoire Naturelle, Paris, France.

    The distalless-related homeogene Dlx5 is expressed in the olfactory placodes and derived tissues and in the anterior-basal forebrain. We investigated the role of Dlx5 in olfactory development. In Dlx5(-/-) mice, the olfactory bulbs (OBs) lack glomeruli, exhibit disorganized cellular layers, and show reduced numbers of TH- and GAD67-positive neurons. The olfactory epithelium in Dlx5(-/-) mice is composed of olfactory receptor neurons (ORNs) that appear identical to wild-type ORNs, but their axons fail to contact the OBs. We transplanted Dlx5(-/-) OBs into a wild-type newborn mouse; wild-type ORN axons enter the mutant OB and form glomeruli, but cannot rescue the lamination defect or the expression of TH and GAD67. Thus, the absence of Dlx5 in the OB does not per se prevent ORN axon ingrowth. In conclusion, Dlx5 plays major roles in the connectivity of ORN axons and in the differentiation of OB interneurons.

    Funded by: Telethon: GP0218Y01, TCP99003

    Molecular and cellular neurosciences 2003;22;4;530-43

  • DLX5 regulates development of peripheral and central components of the olfactory system.

    Long JE, Garel S, Depew MJ, Tobet S and Rubenstein JL

    Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, Langley Porter Psychiatric Institute, University of California, San Francisco, San Francisco, California 94143-0984, USA.

    Induction, neurogenesis, and synaptogenesis of the olfactory bulb are thought to require interactions with the olfactory epithelium. The Dlx family of homeobox genes is expressed in both the olfactory bulb and olfactory epithelium. In particular, Dlx5 is expressed in the olfactory placode, olfactory epithelium, and local circuit neurons of the olfactory bulb. Here we analyzed mice lacking DLX5 function. The Dlx5-/- mutation reduces the size of the olfactory epithelium. Although some olfactory neurons are formed, they fail to generate olfactory axons that innervate the olfactory bulb. Despite the lack of innervation, the olfactory bulb forms, and neurogenesis of projection and local circuit neurons proceeds. However, the mutation has a cell-autonomous effect on the ability of neural progenitors to produce olfactory bulb local circuit neurons, with granule cells more severely affected than periglomerular cells. In addition, the mutation has a noncell-autonomous effect on the morphogenesis of mitral cells.

    Funded by: NIDCR NIH HHS: T32 DE07204; NIMH NIH HHS: K02 MH01046-01, R01 MH49428-01, R01 MH51561-01A1

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;2;568-78

  • Development of the mouse neuromuscular junction in the absence of regulated secretion.

    Heeroma JH, Plomp JJ, Roubos EW and Verhage M

    Department of Molecular Neuroscience, Rudolf Magnus Institute, University of Utrecht Medical Center, Utrecht, The Netherlands.

    To investigate the role of neurotransmitter secretion in the development and stabilization of synapses, the innervation of the diaphragm and intercostal muscles was studied in munc18-1 null mutant mice, which lack regulated secretion. We found that this mutant is completely devoid of both spontaneous and evoked neuromuscular transmission throughout embryonic development. At embryonic day (E) 14, axonal targeting and main branching of the phrenic nerve were normal in this mutant, but tertiary branches were elongated and no terminal branches were observed at this stage, in contrast to control littermates. Acetylcholinesterase staining was observed in the endplate region of mutant muscle from E14 onwards, but not as dense and confined to spots as in controls. Acetylcholine receptor staining was also present in the endplate region of the mutant muscle. In this case, the staining density and the concentration in spots (clusters) were similar to controls, but the distribution of these clusters was less organized. Starting at E15, some receptor clusters co-localized with nerve terminal staining, suggesting synapses, but most clusters remained a-neural. Electron microscopical analysis confirmed the presence of synaptic structures in the mutant. Between E14 and birth, the characteristic staining pattern of nerve branches gradually disappeared in the mutant until, at E18, an elaborate meshwork of nerve fibers with no apparent organization remained. In the same period, most of the motor neuronal cell bodies in the spinal cord degenerated. In contrast, sensory ganglia in the dorsal root showed no obvious degeneration. These data suggest that regulated secretion is not essential for initial axon path finding, clustering of acetylcholine receptors, acetylcholinesterase or the formation of synapses. However, in the absence of regulated secretion, the maintenance of the motor neuronal system, organization of nerve terminal branches and stabilization of synapses is impaired and a-neural postsynaptic elements persist.

    Neuroscience 2003;120;3;733-44

  • A role for neuregulin1 signaling in muscle spindle differentiation.

    Hippenmeyer S, Shneider NA, Birchmeier C, Burden SJ, Jessell TM and Arber S

    Biozentrum, Department of Cell Biology, University of Basel, Klingelbergstrasse 70, 4056-, Basel, Switzerland.

    The maturation of synaptic structures depends on inductive interactions between axons and their prospective targets. One example of such an interaction is the influence of proprioceptive sensory axons on the differentiation of muscle spindles. We have monitored the expression of three transcription factors, Egr3, Pea3, and Erm, that delineate early muscle spindle development in an assay of muscle spindle-inducing signals. We provide genetic evidence that Neuregulin1 (Nrg1) is required for proprioceptive afferent-evoked induction of muscle spindle differentiation in the mouse. Ig-Nrg1 isoforms are preferentially expressed by proprioceptive sensory neurons and are sufficient to induce muscle spindle differentiation in vivo, whereas CRD-Nrg1 isoforms are broadly expressed in sensory and motor neurons but are not required for muscle spindle induction.

    Funded by: NINDS NIH HHS: NS29071, R01 NS027963-13, R01 NS029071-13, R01 NS029071-13S1, R01 NS029071-14, R01 NS029071-15, R01 NS029071-16

    Neuron 2002;36;6;1035-49

  • Gamma protocadherins are required for survival of spinal interneurons.

    Wang X, Weiner JA, Levi S, Craig AM, Bradley A and Sanes JR

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

    The murine genome contains approximately 70 protocadherin (Pcdh) genes. Many are expressed in the nervous system, suggesting that Pcdhs may specify neuronal connectivity. Here, we analyze the 22 contiguous genes of the Pcdh-gamma cluster. Individual neurons express subsets of Pcdh-gamma genes. Pcdh-gamma proteins are present in most neurons and associated with, but not confined to, synapses. Early steps in neuronal migration, axon outgrowth, and synapse formation proceed in mutant mice lacking all 22 Pcdh-gamma genes. At late embryonic stages, however, dramatic neurodegeneration leads to neonatal death. In mutant spinal cord, many interneurons are lost, but sensory and motor neurons are relatively spared. In cultures from mutant spinal cord, neurons differentiate and form synapses but then die. Thus, Pcdh-gamma genes are dispensable for at least some aspects of connectivity but required for survival of specific neuronal types.

    Neuron 2002;36;5;843-54

  • BMP mRNA and protein expression in the developing mouse olfactory system.

    Peretto P, Cummings D, Modena C, Behrens M, Venkatraman G, Fasolo A and Margolis FL

    Department of Human and Animal Biology, University of Torino, 10123 Torino, Italy.

    The bone morphogenetic proteins (BMPs) play fundamental roles during the organization of the central nervous system. The presence of these proteins has also been demonstrated in regions of the adult brain that are characterized by neural plasticity. In this study, we examined the expression of BMP4, 6, and 7 mRNAs and proteins in the murine olfactory system. The olfactory system is a useful model for studying cell proliferation and neural differentiation because both of these processes persist throughout life in the olfactory epithelium (OE) and olfactory bulb (OB). Our results demonstrate a differential expression of BMP4, 6, and 7 in the embryonic, postnatal, and adult olfactory system. In particular, BMP4 and BMP7 showed similar immunostaining patterns, being expressed in the olfactory region from the earliest stages studied (embryonic day 15.5) to adulthood. During development BMPs were expressed in the OE, olfactory bulb nerve layer, glomerular layer (GL), mitral cell layer (MCL), and subventricular zone. During the first postnatal week of life, BMP4 and 7 immunoreactivity (-ir) was particularly evident in the GL, MCL, and in the subependymal layer (SEL), which originates postnatally from the subventricular zone. In adults, BMP4 and 7 immunostaining was present in the GL and SEL. Within the SEL, BMP4 and 7 proteins were expressed primarily in association with the astrocytic glial compartment. BMP6-ir was always found in mature olfactory receptor neurons and their axonal projections to the OB. In summary, these data support the hypothesis that BMPs play a role in the morphogenesis of the olfactory system during development and in its plasticity during adulthood.

    Funded by: NIDCD NIH HHS: DC00347, DC0054

    The Journal of comparative neurology 2002;451;3;267-78

  • Absence of Cajal-Retzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic eminence in Emx1/2 double mutant cerebral cortex.

    Shinozaki K, Miyagi T, Yoshida M, Miyata T, Ogawa M, Aizawa S and Suda Y

    Department of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan.

    Emx1 and Emx2, mouse orthologs of the Drosophila head gap gene, ems, are expressed during corticogenesis. Emx2 null mutants exhibit mild defects in cortical lamination. Segregation of differentiating neurons from proliferative cells is normal for the most part, however, reelin-positive Cajal-Retzius cells are lost by the late embryonic period. Additionally, late-born cortical plate neurons display abnormal position. These types of lamination defects are subtle in the Emx1 mutant cortex. In the present study we show that Emx1 and Emx2 double mutant neocortex is much more severely affected. Thickness of the cerebral wall was diminished with the decrease in cell number. Bromodeoxyuridine uptake in the germinal zone was nearly normal; moreover, no apparent increase in cell death or tetraploid cell number was observed. However, tangential migration of cells from the ganglionic eminence into the neocortex was greatly inhibited. The wild-type ganglionic eminence cells transplanted into Emx1/2-double mutant telencephalon did not move to the cortex. MAP2-positive neuronal bodies and RC2-positive radial glial cells emerged normally, but the laminar structure subsequently formed was completely abnormal. Furthermore, both corticofugal and corticopetal fibers were predominantly absent in the cortex. Most importantly, neither Cajal-Retzius cells nor subplate neurons were found throughout E11.5-E18.5. Thus, this investigation suggests that laminar organization in the cortex or the production of Cajal-Retzius cells and subplate neurons is interrelated to the tangential movement of cells from the ganglionic eminence under the control of Emx1 and Emx2.

    Development (Cambridge, England) 2002;129;14;3479-92

  • Palmitoylcarnitine modulates interaction protein kinase C delta-GAP-43.

    Sobiesiak-Mirska J and Nałecz KA

    Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw, Poland.

    Palmitoylcarnitine, reported previously to promote neuronal differentiation, was observed to affect distribution of protein kinase C (PKC) isoforms in neuroblastoma NB-2a cells, leading to retardation in cytoplasm of high molecular weight species of PKCbeta and delta. Growth cone protein-GAP-43, a PKC substrate, was co-immunoprecipitated with all the conventional and novel PKCs: palmitoylcarnitine, however, decreased its amount exclusively in the complex with PKCdelta. Administration of palmitoylcarnitine, although did not change the subcellular distribution of GAP-43, decreased its phosphorylation, which could regulate other signal transduction pathways (calmodulin and G(0)-dependent).

    Biochemical and biophysical research communications 2002;294;4;823-8

  • Developmental timing of hair follicle and dorsal skin innervation in mice.

    Peters EM, Botchkarev VA, Müller-Röver S, Moll I, Rice FL and Paus R

    Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Hamburg D-20246, Germany.

    The innervation of hair follicles offers an intriguing, yet hardly studied model for the dissection of the stepwise innervation during cutaneous morphogenesis. We have used immunofluorescence and a panel of neuronal markers to characterize the developmental choreography of C57BL/6 mouse backskin innervation. The development of murine skin innervation occurs in successive waves. The first cutaneous nerve fibers appeared before any morphological evidence of hair follicle development at embryonic day 15 (E15). Stage 1 and 2 developing hair follicles were already associated with nerve fibers at E16. These fibers approached a location where later in development the follicular (neural) network A (FNA) is located on fully developed pelage hair follicles. Prior to birth (E18), some nerve fibers had penetrated the epidermis, and an additional set of perifollicular nerve fibers arranged itself around the isthmus and bulge region of stage 5 hair follicles, to develop into the follicular (neural) network B (FNB). By the day of birth (P1), the neuropeptides substance P and calcitonin gene-related peptide became detectable in subcutaneous and dermal nerve fibers first. Newly formed hair follicles on E18 and P1 displayed the same innervation pattern seen in the first wave of hair follicle development. Just prior to epidermal penetration of hair shafts (P5), peptide histidine methionine-IR nerve fibers became detectable and epidermal innervation peaked; such innervation decreased after penetration (P7- P17). Last, tyrosine hydroxylase-IR and neuropeptide Y-IR became readily detectable. This sequence of developing innervation consistently correlates with hair follicle development, indicating a close interdependence of neuronal and epithelial morphogenesis.

    Funded by: NINDS NIH HHS: R0-1 NS34692

    The Journal of comparative neurology 2002;448;1;28-52

  • A point mutant of GAP-43 induces enhanced short-term and long-term hippocampal plasticity.

    Hulo S, Alberi S, Laux T, Muller D and Caroni P

    Neuropharmacology, Centre Médical Universitaire, rue M. Servet 1, 1211 Geneva 4, Switzerland.

    The growth-associated protein GAP-43 (or neuromodulin or B-50) plays a critical role during development in mechanisms of axonal growth and formation of synaptic networks. At later times, GAP-43 has also been implicated in the regulation of synaptic transmission and properties of plasticity such as long-term potentiation. In a molecular approach, we have analyzed transgenic mice overexpressing different mutated forms of GAP-43 or deficient in GAP-43 to investigate the role of the molecule in short-term and long-term plasticity. We report that overexpression of a mutated form of GAP-43 that mimics constitutively phosphorylated GAP-43 results in an enhancement of long-term potentiation in CA1 hippocampal slices. This effect is specific, because LTP was affected neither in transgenic mice overexpressing mutated forms of non-phosphorylatable GAP-43 nor in GAP-43 deficient mice. The increased LTP observed in transgenic mice expressing a constitutively phosphorylated GAP-43 was associated with an increased paired-pulse facilitation as well as an increased summation of responses during high frequency bursts. These results indicate that, while GAP-43 is not necessary for LTP induction, its phosphorylation may regulate presynaptic properties, thereby affecting synaptic plasticity and the induction of LTP.

    The European journal of neuroscience 2002;15;12;1976-82

  • GAP-43 is critical for normal development of the serotonergic innervation in forebrain.

    Donovan SL, Mamounas LA, Andrews AM, Blue ME and McCasland JS

    Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.

    Serotonergic (5-HT) axons from the raphe nuclei are among the earliest afferents to innervate the developing forebrain. The present study examined whether GAP-43, a growth-associated protein expressed on growing 5-HT axons, is necessary for normal 5-HT axonal outgrowth and terminal arborization during the perinatal period. We found a nearly complete failure of 5-HT immunoreactive axons to innervate the cortex and hippocampus in GAP-43-null (GAP43-/-) mice. Abnormal ingrowth of 5-HT axons was apparent on postnatal day 0 (P0); quantitative analysis of P7 brains revealed significant reductions in the density of 5-HT axons in the cortex and hippocampus of GAP43-/- mice relative to wild-type (WT) controls. In contrast, 5-HT axon density was normal in the striatum, septum, and amygdala and dramatically higher than normal in the thalamus of GAP43-/- mice. Concentrations of serotonin and its metabolite, 5-hydroxyindolacetic acid, and norepinephrine were decreased markedly in the anterior and posterior cerebrum but increased in the brainstem of GAP43-/- mice. Cell loss could not account for these abnormalities, because unbiased stereological analysis showed no significant difference in the number of 5-HT dorsal raphe neurons in P7 GAP43-/- versus WT mice. The aberrant 5-HT innervation pattern persisted at P21, indicating a long-term alteration of 5-HT projections to forebrain in the absence of GAP-43. In heterozygotes, the density and morphology of 5-HT axons was intermediate between WT and homozygous GAP43-/- mice. These results suggest that GAP-43 is a key regulator in normal pathfinding and arborization of 5-HT axons during early brain development.

    Funded by: NICHD NIH HHS: HD24061, HD24448, HD24605; NIEHS NIH HHS: ES08131; NINDS NIH HHS: NS31829, NS40779

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;9;3543-52

  • An intrinsic distinction in neuromuscular junction assembly and maintenance in different skeletal muscles.

    Pun S, Sigrist M, Santos AF, Ruegg MA, Sanes JR, Jessell TM, Arber S and Caroni P

    Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.

    We analyzed the formation of neuromuscular junctions (NMJs) in individual muscles of the mouse embryo. Skeletal muscles can be assigned to one of two distinct classes of muscles, termed "Fast Synapsing" (FaSyn) and "Delayed Synapsing" (DeSyn) muscles, which differ significantly with respect to the initial focal clustering of postsynaptic AChRs, the timing of presynaptic maturation, and the maintenance of NMJs in young adult mice. Differences between classes were intrinsic to the muscles and manifested in the absence of innervation or agrin. Paralysis or denervation of young adult muscles resulted in disassembly of AChR clusters on DeSyn muscles, whereas those on FaSyn muscles were preserved. Our results show that postsynaptic differentiation processes intrinsic to FaSyn and DeSyn muscles influence the formation of NMJs during development and their maintenance in the adult.

    Neuron 2002;34;3;357-70

  • Expression of specific glycoconjugates in both primary and secondary olfactory pathways in BALB/C mice.

    Tisay KT, St John JA and Key B

    Department of Anatomy and Developmental Biology, School of Biomedical Sciences, University of Queensland, Brisbane 4072, Australia.

    Binding of cell surface carbohydrates to their receptors specifically promotes axon growth and synaptogenesis in select regions of the developing nervous system. In some cases these interactions depend upon cell-cell adhesion mediated by the same glycoconjugates present on the surface of apposing cells or their processes. We have previously shown that the plant lectin Dolichos biflorus agglutinin (DBA) binds to a subpopulation of mouse primary olfactory neurons whose axons selectively fasciculate prior to terminating in the olfactory bulb. In the present study, we investigated whether these glycoconjugates were also expressed by postsynaptic olfactory neurons specifically within the olfactory pathway. We show here for the first time that DBA ligands were expressed both by a subset of primary olfactory neurons as well as by the postsynaptic mitral/tufted cells in BALB/C mice. These glycoconjugates were first detected on mitral/tufted cell axons during the early postnatal period, at a time when there is considerable synaptogenesis and synaptic remodelling in the primary olfactory cortex. This is one of the few examples of the selective expression of molecules in contiguous axon tracts in the mammalian nervous system. These results suggest that glycoconjugates recognized by DBA may have a specific role in the formation and maintenance of neural connections within a select functional pathway in the brain.

    The Journal of comparative neurology 2002;443;3;213-25

  • Growth-associated protein-43 is required for commissural axon guidance in the developing vertebrate nervous system.

    Shen Y, Mani S, Donovan SL, Schwob JE and Meiri KF

    Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

    Growth-associated protein-43 (GAP-43) is a major growth cone protein whose phosphorylation by PKC in response to extracellular guidance cues can regulate F-actin behavior. Here we show that 100% of homozygote GAP-43 (-/-) mice failed to form the anterior commissure (AC), hippocampal commissure (HC), and corpus callosum (CC) in vivo. Instead, although midline fusion was normal, selective fasciculation between commissural axons was inhibited, and TAG-1-labeled axons tangled bilaterally into Probst's bundles. Moreover, their growth cones had significantly smaller lamellas and reduced levels of F-actin in vitro. Likewise, 100% of GAP-43 (+/-) mice with one disrupted allele also showed defects in HC and CC, whereas the AC was unaffected. Individual GAP-43 (+/-) mice could be assigned to two groups based on the amount that PKC phosphorylation of GAP-43 was reduced in neocortical neurons. In mice with approximately 1% phosphorylation, the HC and CC were absent, whereas in mice with approximately 10% phosphorylation, the HC and CC were smaller. Both results suggest that PKC-mediated signaling in commissural axons may be defective. However, although Probst's bundles formed consistently at the location of the glial wedge, both GAP-43 (-/-) and GAP-43 (+/+) cortical axons were still repulsed by Slit-2 in vitro, precluding failure of this deflective signal from the glial wedge as the source of the phenotype. Nonetheless, the data show that a functional threshold of GAP-43 is required for commissure formation and suggests that failure to regulate F-actin in commissural growth cones may be related to inhibited PKC phosphorylation of GAP-43.

    Funded by: NINDS NIH HHS: NS31829, NS33118

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;1;239-47

  • Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis.

    Washbourne P, Thompson PM, Carta M, Costa ET, Mathews JR, Lopez-Benditó G, Molnár Z, Becher MW, Valenzuela CF, Partridge LD and Wilson MC

    Department of Neurosciences, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131, USA.

    Axon outgrowth during development and neurotransmitter release depends on exocytotic mechanisms, although what protein machinery is common to or differentiates these processes remains unclear. Here we show that the neural t-SNARE (target-membrane-associated-soluble N-ethylmaleimide fusion protein attachment protein (SNAP) receptor) SNAP-25 is not required for nerve growth or stimulus-independent neurotransmitter release, but is essential for evoked synaptic transmission at neuromuscular junctions and central synapses. These results demonstrate that the development of neurotransmission requires the recruitment of a specialized SNARE core complex to meet the demands of regulated exocytosis.

    Funded by: NIMH NIH HHS: MH 4-8989, R01 MH048989

    Nature neuroscience 2002;5;1;19-26

  • The transcription factor Pax6 is required for development of the diencephalic dorsal midline secretory radial glia that form the subcommissural organ.

    Estivill-Torrús G, Vitalis T, Fernández-Llebrez P and Price DJ

    Department of Biomedical Sciences, University of Edinburgh Medical School, Hugh Robson Building, George Square, EH8 9XD, Edinburgh, UK.

    During brain development, Pax6 is expressed in specific regions of the diencephalon including secretory cells of the subcommissural organ (SCO), a circumventricular organ at the forebrain-midbrain boundary that originates from the pretectal dorsal midline neuroepithelial cells beneath the posterior commissure (PC). Homozygous small eye (Sey/Sey) mice lack functional Pax6 protein and fail to develop the SCO, a normal PC and the pineal gland. Small eye heterozygotes (Sey/+) show defective development of the SCO's basal processes which normally penetrate the PC, indicating that normal development of the gland requires normal Pax6 gene-dosage. A correlation between the defects of SCO formation and altered R- and OB-cadherin expression patterns in the SCO is observed in mutants suggesting a role for cadherins in SCO development.

    Mechanisms of development 2001;109;2;215-24

  • 3-Phosphoglycerate dehydrogenase, a key enzyme for l-serine biosynthesis, is preferentially expressed in the radial glia/astrocyte lineage and olfactory ensheathing glia in the mouse brain.

    Yamasaki M, Yamada K, Furuya S, Mitoma J, Hirabayashi Y and Watanabe M

    Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan.

    l-Serine is synthesized from glycolytic intermediate 3-phosphoglycerate and is an indispensable precursor for the synthesis of proteins, membrane lipids, nucleotides, and neuroactive amino acids d-serine and glycine. We have recently shown that l-serine and its interconvertible glycine act as Bergmann glia-derived trophic factors for cerebellar Purkinje cells. To investigate whether such a metabolic neuron-glial relationship is fundamental to the developing and adult brain, we examined by in situ hybridization and immunohistochemistry the cellular expression of 3-phosphoglycerate dehydrogenase (3PGDH), the initial step enzyme for de novo l-serine biosynthesis in animal cells. At early stages when the neural wall consists exclusively of the ventricular zone, neuroepithelial stem cells expressed 3PGDH strongly and homogeneously. Thereafter, 3PGDH expression was downregulated and eventually disappeared in neuronal populations, whereas its high expression was transmitted to the radial glia and later to astrocytes in the gray and white matters. In addition, 3PGDH was highly expressed throughout development in the olfactory ensheathing glia, a specialized supporting cell that thoroughly ensheathes olfactory nerves. These results establish a fundamental link of the radial glia/astrocyte lineage and olfactory ensheathing glia to l-serine biosynthesis in the brain. We discuss this finding in the context of the hypothesis that 3PGDH expression in these glia cells contributes to energy metabolism in differentiating and differentiated neurons and other glia cells, which are known to be vulnerable to energy loss.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;19;7691-704

  • Emx2 directs the development of diencephalon in cooperation with Otx2.

    Suda Y, Hossain ZM, Kobayashi C, Hatano O, Yoshida M, Matsuo I and Aizawa S

    Department of Morphogenesis, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University, Japan.

    The vertebrate brain is among the most complex biological structures of which the organization remains unclear. Increasing numbers of studies have accumulated on the molecular basis of midbrain/hindbrain development, yet relatively little is known about forebrain organization. Nested expression among Otx and Emx genes has implicated their roles in rostral brain regionalization, but single mutant phenotypes of these genes have not provided sufficient information. In order to genetically determine the interaction between Emx and Otx genes in forebrain development, we have examined Emx2(-/-)Otx2(+/-) double mutants and Emx2 knock-in mutants into the Otx2 locus (Otx2(+/Emx2)). Emx2(-/-)Otx2(+/-) double mutants did not develop diencephalic structures such as ventral thalamus, dorsal thalamus/epithalamus and anterior pretectum. The defects were attributed to the loss of the Emx2-positive region at the three- to four-somite stage, when its expression occurs in the laterocaudal forebrain primordia. Ventral structures such as the hypothalamus, mammillary region and tegmentum developed normally. Moreover, dorsally the posterior pretectum and posterior commissure were also present in the double mutants. In contrast, Otx2(+/Emx2) knock-in mutants displayed the majority of these diencephalic structures; however, the posterior pretectum and posterior commissure were specifically absent. Consequently, development of the dorsal and ventral thalamus and anterior pretectum requires cooperation between Emx2 and Otx2, whereas Emx2 expression is incompatible with development of the commissural region of the pretectum.

    Development (Cambridge, England) 2001;128;13;2433-50

  • Failure to express GAP-43 during neurogenesis affects cell cycle regulation and differentiation of neural precursors and stimulates apoptosis of neurons.

    Mani S, Shen Y, Schaefer J and Meiri KF

    Program in Neuroscience, SUNY Upstate Medical University, Syracuse, New York 13210, USA.

    GAP-43 is first expressed in proliferating neuroblasts and is required for maturation of neurons. When GAP-43 is not expressed in differentiating embryonal carcinoma P19 cells, reduced numbers of neurons were generated. Here we show that neuronal differentiation is initially disrupted at the onset of cell-cycle arrest in aggregated, proliferating neuronal precursors. The ratio of nestin:beta-tubulin-labeled progeny generated at this stage suggests that the differentiation is asymmetric. Apoptosis of immature neurons subsequently produced was also significantly induced. In vivo, too, proliferation of neuroblasts was significantly reduced in cortex of GAP-43(-/-) mice at E14.5. These data demonstrate that when GAP-43 is not expressed in proliferating neuroblasts, neural differentiation is not initiated appropriately, inducing apoptosis. Moreover, the concurrent inhibition of Ca2+-dependent adhesion between differentiating P19 cells in aggregates implicates GAP-43 in CAM-mediated signaling during neurogenesis, as has been previously shown in growth cones.

    Funded by: NINDS NIH HHS: NS33118

    Molecular and cellular neurosciences 2001;17;1;54-66

  • Spinal axon regeneration evoked by replacing two growth cone proteins in adult neurons.

    Bomze HM, Bulsara KR, Iskandar BJ, Caroni P and Skene JH

    Cogent Neuroscience, 4425 Ben Franklin Boulevard, Durham, North Carolina 27704, USA.

    In contrast to peripheral nerves, damaged axons in the mammalian brain and spinal cord rarely regenerate. Peripheral nerve injury stimulates neuronal expression of many genes that are not generally induced by CNS lesions, but it is not known which of these genes are required for regeneration. Here we show that co-expressing two major growth cone proteins, GAP-43 and CAP-23, can elicit long axon extension by adult dorsal root ganglion (DRG) neurons in vitro. Moreover, this expression triggers a 60-fold increase in regeneration of DRG axons in adult mice after spinal cord injury in vivo. Replacing key growth cone components, therefore, could be an effective way to stimulate regeneration of CNS axons.

    Nature neuroscience 2001;4;1;38-43

  • Large-scale screen for genes controlling mammalian embryogenesis, using high-throughput gene expression analysis in mouse embryos.

    Neidhardt L, Gasca S, Wertz K, Obermayr F, Worpenberg S, Lehrach H and Herrmann BG

    Max-Planck-Institut für Immunbiologie, Abt. Entwicklungsbiologie, Stübeweg 51, 79108, Freiburg, Germany.

    We have adapted the whole-mount in situ hybridization technique to perform high-throughput gene expression analysis in mouse embryos. A large-scale screen for genes showing specific expression patterns in the mid-gestation embryo was carried out, and a large number of genes controlling development were isolated. From 35760 clones of a 9.5 d.p.c. cDNA library, a total of 5348 cDNAs, enriched for rare transcripts, were selected and analyzed by whole-mount in situ hybridization. Four hundred and twenty-eight clones revealed specific expression patterns in the 9.5 d.p.c. embryo. Of 361 tag-sequenced clones, 198 (55%) represent 154 known mouse genes. Thirty-nine (25%) of the known genes are involved in transcriptional regulation and 33 (21%) in inter- or intracellular signaling. A large number of these genes have been shown to play an important role in embryogenesis. Furthermore, 24 (16%) of the known genes are implicated in human disorders and three others altered in classical mouse mutations. Similar proportions of regulators of embryonic development and candidates for human disorders or mouse mutations are expected among the 163 new mouse genes isolated. Thus, high-throughput gene expression analysis is suitable for isolating regulators of embryonic development on a large-scale, and in the long term, for determining the molecular anatomy of the mouse embryo. This knowledge will provide a basis for the systematic investigation of pattern formation, tissue differentiation and organogenesis in mammals.

    Mechanisms of development 2000;98;1-2;77-94

  • Overexpression of GAP-43 in thalamic projection neurons of transgenic mice does not enable them to regenerate axons through peripheral nerve grafts.

    Mason MR, Campbell G, Caroni P, Anderson PN and Lieberman AR

    Department of Anatomy and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, England.

    It is well established that some populations of neurons of the adult rat central nervous system (CNS) will regenerate axons into a peripheral nerve implant, but others, including most thalamocortical projection neurons, will not. The ability to regenerate axons may depend on whether neurons can express growth-related genes such as GAP-43, whose expression correlates with axon growth during development and with competence to regenerate. Thalamic projection neurons which fail to regenerate into a graft also fail to upregulate GAP-43. We have tested the hypothesis that the absence of strong GAP-43 expression by the thalamic projection neurons prevents them from regenerating their axons, using transgenic mice which overexpress GAP-43. Transgene expression was mapped by in situ hybridization with a digoxigenin-labeled RNA probe and by immunohistochemistry with a monoclonal antibody against the GAP-43 protein produced by the transgene. Many CNS neurons were found to express the mRNA and protein, including neurons of the mediodorsal and ventromedial thalamic nuclei, which rarely regenerate axons into peripheral nerve grafts. Grafts were implanted into the region of these nuclei in the brains of transgenic animals. Although these neurons strongly expressed the transgene mRNA and protein and transported the protein to their axon terminals, they did not regenerate axons into the graft, suggesting that lack of GAP-43 expression is not the only factor preventing thalamocortical neurons regenerating their axons.

    Experimental neurology 2000;165;1;143-52

  • Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray.

    Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X, Grahovac MJ, Pantano S, Sano Y, Piao Y, Nagaraja R, Doi H, Wood WH, Becker KG and Ko MS

    Laboratory of Genetics and DNA Array Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224-6820, USA.

    cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;16;9127-32

  • Absence of GAP-43 can protect neurons from death.

    Gagliardini V, Dusart I and Fankhauser C

    Brain Research Institute, Department of Neuromorphology, University of Zurich and Swiss Federal Institute of Technology Zurich, Winterthurerstrasse 190, Zurich, 8057, Switzerland.

    The main function of GAP-43 is thought to be regulating growth cone motility and axon guidance signals. GAP-43 is highly expressed during development and in regenerating nerves and in particular regions of the adult brain. We here present the first evidence that GAP-43 can modulate guidance signals emanating from Semaphorin III (SemaIII) in cultured NGF-dependent sensory neurons. We further show that absence of GAP-43 dramatically increases resistance of specific sensory neurons to apoptotic stimuli in vitro. NGF-dependent sensory neurons from GAP-43 (+/-) and null mutant mice are strongly protected against SemaIII-induced death. Furthermore, NGF- and BDNF-dependent neurons, but not NT-3-dependent neurons, from GAP-43 null mutant mice are much more resistant to apoptosis induced by trophic factor deprivation. We also show that early postnatal Purkinje cells from GAP-43 (+/-) mice are more resistant to cell death in organotypic cultures. We conclude that GAP-43 can influence neuronal survival and modulate repulsive axon guidance signals.

    Molecular and cellular neurosciences 2000;16;1;27-33

  • GAP43, MARCKS, and CAP23 modulate PI(4,5)P(2) at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism.

    Laux T, Fukami K, Thelen M, Golub T, Frey D and Caroni P

    Friedrich Miescher Institute, CH-4058 Basel, Switzerland.

    The dynamic properties of the cell cortex and its actin cytoskeleton determine important aspects of cell behavior and are a major target of cell regulation. GAP43, myristoylated alanine-rich C kinase substrate (MARCKS), and CAP23 (GMC) are locally abundant, plasmalemma-associated PKC substrates that affect actin cytoskeleton. Their expression correlates with morphogenic processes and cell motility, but their role in cortex regulation has been difficult to define mechanistically. We now show that the three proteins accumulate at rafts, where they codistribute with PI(4,5)P(2), and promote its retention and clustering. Binding and modulation of PI(4, 5)P(2) depended on the basic effector domain (ED) of these proteins, and constructs lacking the ED functioned as dominant inhibitors of plasmalemmal PI(4,5)P(2) modulation. In the neuron-like cell line, PC12, NGF- and substrate-induced peripheral actin structures, and neurite outgrowth were greatly augmented by any of the three proteins, and suppressed by DeltaED mutants. Agents that globally mask PI(4,5)P(2) mimicked the effects of GMC on peripheral actin recruitment and cell spreading, but interfered with polarization and process formation. Dominant negative GAP43(DeltaED) also interfered with peripheral nerve regeneration, stimulus-induced nerve sprouting and control of anatomical plasticity at the neuromuscular junction of transgenic mice. These results suggest that GMC are functionally and mechanistically related PI(4,5)P(2) modulating proteins, upstream of actin and cell cortex dynamics regulation.

    The Journal of cell biology 2000;149;7;1455-72

  • Shared and unique roles of CAP23 and GAP43 in actin regulation, neurite outgrowth, and anatomical plasticity.

    Frey D, Laux T, Xu L, Schneider C and Caroni P

    Friedrich Miescher Institute, CH-4058 Basel, Switzerland.

    CAP23 is a major cortical cytoskeleton-associated and calmodulin binding protein that is widely and abundantly expressed during development, maintained in selected brain structures in the adult, and reinduced during nerve regeneration. Overexpression of CAP23 in adult neurons of transgenic mice promotes nerve sprouting, but the role of this protein in process outgrowth was not clear. Here, we show that CAP23 is functionally related to GAP43, and plays a critical role to regulate nerve sprouting and the actin cytoskeleton. Knockout mice lacking CAP23 exhibited a pronounced and complex phenotype, including a defect to produce stimulus-induced nerve sprouting at the adult neuromuscular junction. This sprouting deficit was rescued by transgenic overexpression of either CAP23 or GAP43 in adult motoneurons. Knockin mice expressing GAP43 instead of CAP23 were essentially normal, indicating that, although these proteins do not share homologous sequences, GAP43 can functionally substitute for CAP23 in vivo. Cultured sensory neurons lacking CAP23 exhibited striking alterations in neurite outgrowth that were phenocopied by low doses of cytochalasin D. A detailed analysis of such cultures revealed common and unique functions of CAP23 and GAP43 on the actin cytoskeleton and neurite outgrowth. The results provide compelling experimental evidence for the notion that CAP23 and GAP43 are functionally related intrinsic determinants of anatomical plasticity, and suggest that these proteins function by locally promoting subplasmalemmal actin cytoskeleton accumulation.

    The Journal of cell biology 2000;149;7;1443-54

  • ETS gene Er81 controls the formation of functional connections between group Ia sensory afferents and motor neurons.

    Arber S, Ladle DR, Lin JH, Frank E and Jessell TM

    Department of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Columbia University, New York, New York 10032, USA.

    The connections formed between sensory and motor neurons (MNs) play a critical role in the control of motor behavior. During development, the axons of proprioceptive sensory neurons project into the spinal cord and form both direct and indirect connections with MNs. Two ETS transcription factors, ER81 and PEA3, are expressed by developing proprioceptive neurons and MNs, raising the possibility that these genes are involved in the formation of sensory-motor connections. Er81 mutant mice exhibit a severe motor discoordination, yet the specification of MNs and induction of muscle spindles occurs normally. The motor defect in Er81 mutants results from a failure of group Ia proprioceptive afferents to form a discrete termination zone in the ventral spinal cord. As a consequence there is a dramatic reduction in the formation of direct connections between proprioceptive afferents and MNs. ER81 therefore controls a late step in the establishment of functional sensory-motor circuitry in the developing spinal cord.

    Cell 2000;101;5;485-98

  • GAP-43 mediates retinal axon interaction with lateral diencephalon cells during optic tract formation.

    Zhang F, Lu C, Severin C and Sretavan DW

    Beckman Vision Center, Neuroscience and Biomedical Sciences Programs, Departments of Ophthalmology & Physiology, University of California, San Francisco, USA.

    GAP-43 is an abundant intracellular growth cone protein that can serve as a PKC substrate and regulate calmodulin availability. In mice with targeted disruption of the GAP-43 gene, retinal ganglion cell (RGC) axons fail to progress normally from the optic chiasm into the optic tracts. The underlying cause is unknown but, in principle, can result from either the disruption of guidance mechanisms that mediate axon exit from the midline chiasm region or defects in growth cone signaling required for entry into the lateral diencephalic wall to form the optic tracts. Results here show that, compared to wild-type RGC axons, GAP-43-deficient axons exhibit reduced growth in the presence of lateral diencephalon cell membranes. Reduced growth is not observed when GAP-43-deficient axons are cultured with optic chiasm, cortical, or dorsal midbrain cells. Lateral diencephalon cell conditioned medium inhibits growth of both wild-type and GAP-43-deficient axons to a similar extent and does not affect GAP-43-deficient axons more so. Removal or transplant replacement of the lateral diencephalon optic tract entry zone in GAP-43-deficient embryo preparations results in robust RGC axon exit from the chiasm. Together these data show that RGC axon exit from the midline region does not require GAP-43 function. Instead, GAP-43 appears to mediate RGC axon interaction with guidance cues in the lateral diencephalic wall, suggesting possible involvement of PKC and calmodulin signaling during optic tract formation.

    Funded by: NEI NIH HHS: EY02162, EY10688; NINDS NIH HHS: NSO 7067

    Development (Cambridge, England) 2000;127;5;969-80

  • Synaptic assembly of the brain in the absence of neurotransmitter secretion.

    Verhage M, Maia AS, Plomp JJ, Brussaard AB, Heeroma JH, Vermeer H, Toonen RF, Hammer RE, van den Berg TK, Missler M, Geuze HJ and Südhof TC

    Molecular Neuroscience, Rudolf Magnus Institute, University of Utrecht Medical Centre, Utrecht, Netherlands.

    Brain function requires precisely orchestrated connectivity between neurons. Establishment of these connections is believed to require signals secreted from outgrowing axons, followed by synapse formation between selected neurons. Deletion of a single protein, Munc18-1, in mice leads to a complete loss of neurotransmitter secretion from synaptic vesicles throughout development. However, this does not prevent normal brain assembly, including formation of layered structures, fiber pathways, and morphologically defined synapses. After assembly is completed, neurons undergo apoptosis, leading to widespread neurodegeneration. Thus, synaptic connectivity does not depend on neurotransmitter secretion, but its maintenance does. Neurotransmitter secretion probably functions to validate already established synaptic connections.

    Science (New York, N.Y.) 2000;287;5454;864-9

  • Assignment of microrchidia (Morc) to mouse chromosome 16 by interspecific backcross linkage analysis and human chromosome 3q13 using somatic cell hybrids and in situ hybridization.

    Inoue N, Wei F, Seldin MF, Zinn AR and Watson ML

    Eugene McDermott Center for Human Growth and Development and Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas TX, USA.

    Funded by: NHGRI NIH HHS: HG00734

    Cytogenetics and cell genetics 2000;90;1-2;123-5

  • Transgenic expression of B-50/GAP-43 in mature olfactory neurons triggers downregulation of native B-50/GAP-43 expression in immature olfactory neurons.

    Holtmaat AJ, Huizinga CT, Margolis FL, Gispen WH and Verhaagen J

    Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ, Amsterdam, The Netherlands.

    The adult mammalian olfactory neuroepithelium is an unusual neural tissue, since it maintains its capacity to form new neurons throughout life. Newly formed neurons differentiate in the basal layers of the olfactory neuroepithelium and express B-50/GAP-43, a protein implicated in neurite outgrowth. During maturation these neurons migrate into the upper portion of the epithelium, upregulate expression of olfactory marker protein (OMP) and concomitantly downregulate the expression of B-50/GAP-43. Transgenic mice that exhibit OMP-promoter directed expression of B-50/GAP-43 in mature olfactory neurons display an unexpected decrease in the complement of B-50/GAP-43-positive cells in the lower region of the olfactory epithelium [A.J.G.D. Holtmaat, P.A. Dijkhuizen, A.B. Oestreicher, H. J. Romijn, N.M.T. Van der Lugt, A. Berns, F.L. Margolis, W.H. Gispen, J. Verhaagen, Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular growth, J. Neurosci. 15 (1995) 7953-7965]. We have investigated whether the decrement in B-50/GAP-43-positive cells in this region was due to a dislocation of the immature neurons to other regions of the olfactory epithelium or to a downregulation of B-50/GAP-43 synthesis in these immature neurons. In eight of nine independent transgenic mouse lines that express the transgene in different numbers of olfactory neurons, a decline in the number of B-50/GAP-43-expressing neurons in the basal portion of the olfactory neuroepithelium was observed, both at the protein level and the mRNA level. An alternative marker for immature cells, a juvenile form of tubulin, was normally expressed in this location, indicating that the olfactory epithelium of OMP-B-50/GAP-43 transgenic mice contains a normal complement of immature olfactory neurons and that most of these neurons display a downregulation of B-50/GAP-43 expression.

    Brain research. Molecular brain research 1999;74;1-2;197-207

  • Peripheral nervous system defects in erbB2 mutants following genetic rescue of heart development.

    Woldeyesus MT, Britsch S, Riethmacher D, Xu L, Sonnenberg-Riethmacher E, Abou-Rebyeh F, Harvey R, Caroni P and Birchmeier C

    Max-Delbrück-Center for Molecular Medicine, 13092 Berlin, Germany.

    The ErbB2 tyrosine kinase functions as coreceptor for the neuregulin receptors ErbB3 and ErbB4 and can participate in signaling of EGF receptor (ErbB1), interleukin receptor gp130, and G-protein coupled receptors. ErbB2(-/-) mice die at midgestation because of heart malformation. Here, we report a genetic rescue of their heart development by myocardial expression of erbB2 cDNA that allows survival of the mutants to birth. In rescued erbB2 mutants, Schwann cells are lacking. Motoneurons form and can project to muscle, but nerves are poorly fasciculated and disorganized. Neuromuscular junctions form, as reflected in clustering of AChR and postsynaptic expression of the genes encoding the alpha-AChR, AChE, epsilon-AChR, and the RI subunit of the cAMP protein kinase. However, a severe loss of motoneurons on cervical and lumbar, but not on thoracic levels occurs. Our results define the roles of Schwann cells during motoneuron and synapse development, and reveal different survival requirements for distinct motoneuron populations.

    Genes & development 1999;13;19;2538-48

  • Genetic mapping of six mouse peroxiredoxin genes and fourteen peroxiredoxin related sequences.

    Lyu MS, Rhee SG, Chae HZ, Lee TH, Adamson MC, Kang SW, Jin DY, Jeang KT and Kozak CA

    Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, NIH, Building 4, Room 329, 4 Center Drive MSC 0460, Bethesda, MD 20892-0460, USA.

    Mammalian genome : official journal of the International Mammalian Genome Society 1999;10;10;1017-9

  • A candidate target for G protein action in brain.

    Chen LT, Gilman AG and Kozasa T

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.

    An effector candidate for G protein action, GRIN1, was identified by screening a cDNA expression library with phosphorylated GTPgammaS-G(z)alpha as a probe. GRIN1 is a novel protein without substantial homology to known protein domains. It is expressed largely in brain and binds specifically to activated G(z)alpha, G(o)alpha, and G(i)alpha through its carboxyl-terminal region. The protein KIAA0514 (GRIN2) is homologous to GRIN1 at its carboxyl terminus and also binds to activated G(o)alpha. Both GRIN1 and G(o)alpha are membrane-bound proteins that are enriched in the growth cones of neurites. Coexpression of GRIN1 or GRIN2 with activated G(o)alpha causes formation of a network of fine processes in Neuro2a cells, suggesting that these pathways may function downstream of G(o)alpha to control growth of neurites.

    Funded by: NIGMS NIH HHS: GM34497

    The Journal of biological chemistry 1999;274;38;26931-8

  • Identification and genetic mapping of differentially expressed genes in mice differing at the If1 interferon regulatory locus.

    Kozak CA, Su Y, Raj NB and Pitha PM

    Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Building 4, Room 329, 4 Center Drive MSC 0460, Bethesda, Maryland 20982-0460, USA.

    A subtractive cDNA library was used to identify differentially expressed genes in mouse strains that differ at If1, a locus that regulates response to interferon induction by Newcastle Disease Virus infection. Among the isolated clones, sequence analysis identified the ribosomal proteins L37a and S8 as well as cDNAs for thymosine beta4, the QM transcriptional factor, and a novel genetic sequence. Analysis of two multilocus mouse crosses showed that the thymosine beta4 gene, Ptmb4, is present as a single-copy gene that maps to distal Chr X. The L37a, S8, and QM clones are all members of large multilocus families. These five clones were used to determine the map locations for 37 loci, of which 31 had not previously been described. The novel genetic sequence, D3Ppr1, mapped to distal Chr 3 near the position of the If1 locus, suggesting it may be a candidate for this regulatory gene.

    Funded by: NIAID NIH HHS: AI19737

    Mammalian genome : official journal of the International Mammalian Genome Society 1999;10;9;853-7

  • Disrupted cortical map and absence of cortical barrels in growth-associated protein (GAP)-43 knockout mice.

    Maier DL, Mani S, Donovan SL, Soppet D, Tessarollo L, McCasland JS and Meiri KF

    Department of Anatomy and Cell Biology, State University of New York Health Science Center, Syracuse, NY 13210, USA.

    There is strong evidence that growth-associated protein (GAP-43), a protein found only in the nervous system, regulates the response of neurons to axonal guidance signals. However, its role in complex spatial patterning in cerebral cortex has not been explored. We show that mice lacking GAP-43 expression (-/-) fail to establish the ordered whisker representation (barrel array) normally found in layer IV of rodent primary somatosensory cortex. Thalamocortical afferents to -/- cortex form irregular patches in layer IV within a poorly defined cortical field, which varies between hemispheres, rather than the stereotypic, whisker-specific, segregated map seen in normal animals. Furthermore, many thalamocortical afferents project abnormally to widely separated cortical targets. Taken together, our findings indicate a loss of identifiable whisker territories in the GAP-43 -/- mouse cortex. Here, we present a disrupted somatotopic map phenotype in cortex, in clear contrast to the blurring of boundaries within an ordered whisker map in other barrelless mutants. Our results indicate that GAP-43 expression is critical for the normal establishment of ordered topography in barrel cortex.

    Funded by: NINDS NIH HHS: NS31829, NS33118, R01 NS033118

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;16;9397-402

  • Requirement for the homeobox gene Hb9 in the consolidation of motor neuron identity.

    Arber S, Han B, Mendelsohn M, Smith M, Jessell TM and Sockanathan S

    Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Columbia University, New York, New York 10032, USA.

    The homeobox gene Hb9, like its close relative MNR2, is expressed selectively by motor neurons (MNs) in the developing vertebrate CNS. In embryonic chick spinal cord, the ectopic expression of MNR2 or Hb9 is sufficient to trigger MN differentiation and to repress the differentiation of an adjacent population of V2 interneurons. Here, we provide genetic evidence that Hb9 has an essential role in MN differentiation. In mice lacking Hb9 function, MNs are generated on schedule and in normal numbers but transiently acquire molecular features of V2 interneurons. The aberrant specification of MN identity is associated with defects in the migration of MNs, the emergence of the subtype identities of MNs, and the projection of motor axons. These findings show that HB9 has an essential function in consolidating the identity of postmitotic MNs.

    Neuron 1999;23;4;659-74

  • Schwann cell-derived Desert hedgehog controls the development of peripheral nerve sheaths.

    Parmantier E, Lynn B, Lawson D, Turmaine M, Namini SS, Chakrabarti L, McMahon AP, Jessen KR and Mirsky R

    Department of Anatomy and Developmental Biology, University College London, United Kingdom.

    We show that Schwann cell-derived Desert hedgehog (Dhh) signals the formation of the connective tissue sheath around peripheral nerves. mRNAs for dhh and its receptor patched (ptc) are expressed in Schwann cells and perineural mesenchyme, respectively. In dhh-/- mice, epineurial collagen is reduced, while the perineurium is thin and disorganized, has patchy basal lamina, and fails to express connexin 43. Perineurial tight junctions are abnormal and allow the passage of proteins and neutrophils. In nerve fibroblasts, Dhh upregulates ptc and hedgehog-interacting protein (hip). These experiments reveal a novel developmental signaling pathway between glia and mesenchymal connective tissue and demonstrate its molecular identity in peripheral nerve. They also show that Schwann cell-derived signals can act as important regulators of nerve development.

    Funded by: Wellcome Trust

    Neuron 1999;23;4;713-24

  • Analysis of mice carrying targeted mutations of the glucocorticoid receptor gene argues against an essential role of glucocorticoid signalling for generating adrenal chromaffin cells.

    Finotto S, Krieglstein K, Schober A, Deimling F, Lindner K, Brühl B, Beier K, Metz J, Garcia-Arraras JE, Roig-Lopez JL, Monaghan P, Schmid W, Cole TJ, Kellendonk C, Tronche F, Schütz G and Unsicker K

    Neuroanatomy, University of Heidelberg, D-69120 Heidelberg, Germany. klaus.unsicker@urz.uni-heidelberg.de

    Molecular mechanisms underlying the generation of distinct cell phenotypes is a key issue in developmental biology. A major paradigm of determination of neural cell fate concerns the development of sympathetic neurones and neuroendocrine chromaffin cells from a common sympathoadrenal (SA) progenitor cell. Two decades of in vitro experiments have suggested an essential role of glucocorticoid receptor (GR)-mediated signalling in generating chromaffin cells. Targeted mutation of the GR should consequently abolish chromaffin cells. The present analysis of mice lacking GR gene product demonstrates that animals have normal numbers of adrenal chromaffin cells. Moreover, there are no differences in terms of apoptosis and proliferation or in expression of several markers (e.g. GAP43, acetylcholinesterase, adhesion molecule L1) of chromaffin cells in GR-deficient and wild-type mice. However, GR mutant mice lack the adrenaline-synthesizing enzyme PNMT and secretogranin II. Chromaffin cells of GR-deficient mice exhibit the typical ultrastructural features of this cell phenotype, including the large chromaffin granules that distinguish them from sympathetic neurones. Peripherin, an intermediate filament of sympathetic neurones, is undetectable in chromaffin cells of GR mutants. Finally, when stimulated with nerve growth factor in vitro, identical proportions of chromaffin cells from GR-deficient and wild-type mice extend neuritic processes. We conclude that important phenotypic features of chromaffin cells that distinguish them from sympathetic neurones develop normally in the absence of GR-mediated signalling. Most importantly, chromaffin cells in GR-deficient mice do not convert to a neuronal phenotype. These data strongly suggest that the dogma of an essential role of glucocorticoid signalling for the development of chromaffin cells must be abandoned.

    Development (Cambridge, England) 1999;126;13;2935-44

  • Overexpression of GAP-43 induces prolonged sprouting and causes death of adult motoneurons.

    Harding DI, Greensmith L, Mason M, Anderson PN and Vrbová G

    Department of Anatomy, University College London, UK.

    In neurodegenerative diseases, neurons undergo prolonged periods of sprouting. Whether this sprouting compromises these neurons is unknown. Here, we examined the effect of axotomy on adult motoneurons undergoing prolonged sprouting in transgenic mice that overexpress GAP-43 (growth-associated protein). Sciatic nerve injury in these adult mice results in motoneuron death, but has no effect in non-transgenic mice. Thus, continued growth of motor axons renders adult motoneurons susceptible to nerve injury and compromises their long-term survival. The progressive nature of neurodegenerative diseases may therefore be caused by prolonged sprouting.

    Funded by: Wellcome Trust

    The European journal of neuroscience 1999;11;7;2237-42

  • Role of brain-derived neurotrophic factor in target invasion in the gustatory system.

    Ringstedt T, Ibáñez CF and Nosrat CA

    Laboratory of Molecular Neurobiology, Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden.

    Brain-derived neurotrophic factor (BDNF) is a survival factor for different classes of neurons, including gustatory neurons. We have studied innervation and development of the gustatory system in transgenic mice overexpressing BDNF under the control of regulatory sequences from the nestin gene, an intermediate filament gene expressed in precursor cells of the developing nervous system and muscle. In transgenic mice, the number and size of gustatory papillae were decreased, circumvallate papillae had a deranged morphology, and there was also a severe loss of lingual taste buds. Paradoxically, similar deficits have been found in BDNF knock-out mice, which lack gustatory neurons. However, the number of neurons in gustatory ganglia was increased in BDNF-overproducing mice. Although gustatory fibers reached the tongue in normal numbers, the amount and density of nerve fibers in gustatory papillae were reduced in transgenic mice compared with wild-type littermates. Gustatory fibers appeared stalled at the base of the tongue, a site of ectopic BDNF expression, where they formed abnormal branches and sprouts. Interestingly, palatal taste buds, which are innervated by gustatory neurons whose afferents do not traverse sites of ectopic BDNF expression, appeared unaffected. We suggest that lingual gustatory deficits in BDNF overexpressing mice are a consequence of the failure of their BDNF-dependent afferents to reach their targets because of the effects of ectopically expressed BDNF on fiber growth. Our findings suggest that mammalian taste buds and gustatory papillae require proper BDNF-dependent gustatory innervation for development and that the correct spatial expression of BDNF in the tongue epithelium is crucial for appropriate target invasion and innervation.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;9;3507-18

  • A key role for GAP-43 in the retinotectal topographic organization.

    Zhu Q and Julien JP

    Centre for Research in Neuroscience, McGill University, The Montreal General Hospital Research Institute, Montreal, H3G 1A4, Canada.

    To have a proper spatial visual perception, vertebrate retinal ganglion cells connect to their brain targets in a highly ordered fashion. The molecular bases for such topographic retinotectal connection in mammals still remain largely unknown. Using the gene knock-out approach in mice, we report here a key role for the GAP-43 growth cone protein in the development of the visual system. In mice bearing a targeted disruption of GAP-43 exon 1, a high proportion of retinal ganglion cell (RGC) axons was found to grow abnormally into the ipsilateral optic tract and into the hypothalamus. After leaving the optic chiasm during development, the GAP-43-deficient RGC axons generally follow the optic tracts but are unable to form proper terminal zones in the lateral geniculate nucleus. Moreover, in the superior colliculus, RGC axons lacking GAP-43 are intermingled. These results suggest an essential role for GAP-43 in development of the topographic retinotectal connection.

    Experimental neurology 1999;155;2;228-42

  • Excitatory synaptic transmission and its modulation by PKC is unchanged in the hippocampus of GAP-43-deficient mice.

    Capogna M, Fankhauser C, Gagliardini V, Gähwiler BH and Thompson SM

    Brain Research Institute, University of Zurich, Switzerland.

    We compared excitatory synaptic transmission between hippocampal pyramidal cells in dissociated hippocampal cell cultures and in area CA3 of hippocampal slice cultures derived from wild-type mice and mice with a genetic deletion of the presynaptic growth associated protein GAP-43. The basal frequency and amplitude of action potential-dependent and -independent spontaneous excitatory postsynaptic currents were similar in both groups. The probability that any two CA3 pyramidal cells in wild-type or GAP-43 knockout (-/-) slice cultures were synaptically connected was assessed with paired recordings and was not different. Furthermore, unitary synaptic responses were similar in the two genotypes. Bath application of phorbol 12,13-diacetate (0.6-3 microM) elicited a comparable increase in the frequency of miniature excitatory synaptic currents in wild-type and GAP-43 (-/-) cultures. This effect was blocked by the protein kinase C inhibitor, bisindolylmaleimide I (1.2 microM). Finally, 3 microM phorbol 12,13-diacetate potentiated the amplitude of unitary synaptic currents to a comparable extent in wild-type and GAP-43 (-/-) slice cultures. We conclude that GAP-43 is not required for normal excitatory synaptic transmission or the potentiation of presynaptic glutamate release mediated by activation of protein kinase C in the hippocampus.

    The European journal of neuroscience 1999;11;2;433-40

  • Substrate phosphorylation in the protein kinase Cgamma knockout mouse.

    Ramakers GM, Gerendasy DD and de Graan PN

    Rudolf Magnus Institute for Neurosciences, Department of Medical Pharmacology, Unversiteitsweg 100, 3584 CG Utrecht, The Netherlands. geert.ramakers@basalmed.uio.no

    The phosphorylation state of three identified neural-specific protein kinase C substrates (RC3, GAP-43/B-50, and MARCKS) was monitored in hippocampal slices of mice lacking the gamma-subtype of protein kinase C and wild-type controls by quantitative immunoprecipitation following 32Pi labeling. Depolarization with potassium, activation of glutamate receptors with glutamate, or direct stimulation of protein kinase C with a phorbol ester increased RC3 phosphorylation in wild-type animals but failed to affect RC3 phosphorylation in mice lacking the gamma-subtype of protein kinase C. Our results suggests the following biochemical pathway: activation of a postsynaptic (metabotropic) glutamate receptor stimulates the gamma-subtype of protein kinase C, which in turn phosphorylates RC3. The inability to increase RC3 phosphorylation in mice lacking the gamma-subtype of protein kinase C by membrane depolarization or glutamate receptor activation may contribute to the spatial learning deficits and impaired hippocampal LTP observed in these mice.

    Funded by: NIGMS NIH HHS: GM-32355; NINDS NIH HHS: NS-35831

    The Journal of biological chemistry 1999;274;4;1873-4

  • Randomized retinal ganglion cell axon routing at the optic chiasm of GAP-43-deficient mice: association with midline recrossing and lack of normal ipsilateral axon turning.

    Sretavan DW and Kruger K

    Departments of Ophthalmology and Physiology, University of California San Francisco, San Francisco, California 94143, USA.

    During mammalian development, retinal ganglion cell (RGC) axons from nasal retina cross the optic chiasm midline, whereas temporal retina axons do not and grow ipsilaterally, resulting in a projection of part of the visual world onto one side of the brain while the remaining part is represented on the opposite side. Previous studies have shown that RGC axons in GAP-43-deficient mice initially fail to grow from the optic chiasm to form optic tracts and are delayed temporarily in the midline region. Here we show that this delayed RGC axon exit from the chiasm is characterized by abnormal randomized axon routing into the ipsilateral and contralateral optic tracts, leading to duplicated representations of the visual world in both sides of the brain. Within the chiasm, individual contralaterally projecting axons grow in unusual semicircular trajectories, and the normal ipsilateral turning of ventral temporal axons is absent. These effects on both axon populations suggest that GAP-43 does not mediate pathfinding specifically for one or the other axon population but is more consistent with a model in which the initial pathfinding defect at the chiasm/tract transition zone leads to axons backing up into the chiasm, resulting in circular trajectories and eventual random axon exit into one or the other optic tract. Unusual RGC axon trajectories include chiasm midline recrossing similar to abnormal CNS midline recrossing in invertebrate "roundabout" mutants and Drosophila with altered calmodulin function. This resemblance and the fact that GAP-43 also has been proposed to regulate calmodulin availability raise the possibility that calmodulin function is involved in CNS midline axon guidance in both vertebrates and invertebrates.

    Funded by: NEI NIH HHS: EY 02162, EY 10688

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;24;10502-13

  • Expression of Kv1.1, a Shaker-like potassium channel, is temporally regulated in embryonic neurons and glia.

    Hallows JL and Tempel BL

    The Virginia Merrill Bloedel Hearing Research Center and the Departments of Pharmacology and Otolaryngology-Head and Neck Surgery, University of Washington School of Medicine, Seattle, Washington 98195, USA.

    Kv1.1, a Shaker-like voltage-gated potassium channel, is strongly expressed in a variety of neurons in adult rodents, in which it appears to be involved in regulating neuronal excitability. Here we show that Kv1.1 is also expressed during embryonic development in the mouse, exhibiting two transient peaks of expression around embryonic day 9.5 (E9.5) and E14.5. Using both in situ hybridization and immunocytochemistry, we have identified several cell types and tissues that express Kv1.1 RNA and protein. At E9.5, Kv1.1 RNA and protein are detected transiently in non-neuronal cells in several regions of the early CNS, including rhombomeres 3 and 5 and ventricular zones in the mesencephalon and diencephalon. At E14.5, several cell types in both the CNS and peripheral nervous system express Kv1.1, including neuronal cells (sensory ganglia and outer aspect of cerebral hemispheres) and glial cells (radial glia, satellite cells, and Schwann cell precursors). These data show that Kv1.1 is expressed transiently in a variety of neuronal and non-neuronal cells during restricted periods of embryonic development. Although the functional roles of Kv1.1 in development are not understood, the cell-specific localization and timing of expression suggest this channel may play a role in several developmental processes, including proliferation, migration, or cell-cell adhesion.

    Funded by: NICHD NIH HHS: N01-HD-6-2915; NIGMS NIH HHS: GM07750; NINDS NIH HHS: NS27206

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;15;5682-91

  • Retinal ganglion cell axon progression from the optic chiasm to initiate optic tract development requires cell autonomous function of GAP-43.

    Kruger K, Tam AS, Lu C and Sretavan DW

    Departments of Ophthalmology and Physiology, University of California, San Francisco, California 94143, USA.

    Pathfinding mechanisms underlying retinal ganglion cell (RGC) axon growth from the optic chiasm into the optic tract are unknown. Previous work has shown that mouse embryos deficient in GAP-43 have an enlarged optic chiasm within which RGC axons were reportedly stalled. Here we have found that the enlarged chiasm of GAP-43 null mouse embryos appears subsequent to a failure of the earliest RGC axons to progress laterally through the chiasm-tract transition zone to form the optic tract. Previous work has shown that ventral diencephalon CD44/stage-specific embryonic antigen (SSEA) neurons provide guidance information for RGC axons during chiasm formation. Here we found that in the chiasm-tract transition zone, axons of CD44/SSEA neurons precede RGC axons into the lateral diencephalic wall and like RGC axons also express GAP-43. However unlike RGC axons, CD44/SSEA axon trajectories are unaffected in GAP-43 null embryos, indicating that GAP-43-dependent guidance at this site is RGC axon specific or occurs only at specific developmental times. To determine whether the phenotype results from loss of GAP-43 in RGCs or in diencephalon components such as CD44/SSEA axons, wild-type, heterozygous, or homozygous GAP-43 null donor retinal tissues were grafted onto host diencephalons of all three genotypes, and graft axon growth into the optic tract region was assessed. Results show that optic tract development requires cell autonomous GAP-43 function in RGC axons and not in cellular elements of the ventral diencephalon or transition zone.

    Funded by: NEI NIH HHS: EY02162, EY10688

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;15;5692-705

  • p59fyn and pp60c-src modulate axonal guidance in the developing mouse olfactory pathway.

    Morse WR, Whitesides JG, LaMantia AS and Maness PF

    Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599, USA.

    The Src-family tyrosine kinases p59fyn and pp60c-src are localized on axons of the mouse olfactory nerve during the initial stages of axonal growth, but their functional roles remain to be defined. To study the role of these kinases, we analyzed the trajectory of the olfactory nerve in E11.5 homozygous null mutant mice lacking single src or fyn gens and double mutants lacking both genes. Primary olfactory axons of single and double mutants exited the olfactory epithelium and projected toward the telencephalon, but displayed differences in fasciculation. The fyn-minus olfactory nerve had significantly more fascicles than than src-minus nerve. Most strikingly, the primary olfactory nerve of src/fyn double mutants showed the greatest degree of defasciculation. These defects, identified by NCAM labeling, were not due to apparent changes in the size of the olfactory epithelium. With the exception of the src-minus mice, which had fever fascicles than the wild type, no obvious differences were observed in coalescence of vomeronasal axons from mutant mice. The mesenchyme of the double and single mutants exhibited only subtle changes in laminin and fibronectin staining, indicating that the adhesive environment of the mesenchyme may contribute in part to defects in fasciculation. The results suggest that signaling pathways mediated by p59fyn and pp60c-src contribute to the appropriate fasciculation of axons in the nascent olfactory system, and comprise partially compensatory mechanisms for axonal adhesion and guidance.

    Journal of neurobiology 1998;36;1;53-63

  • Characterization and localization of Mox2, the gene encoding the murine homolog of the rat MRC OX-2 membrane glycoprotein.

    Borriello F, Tizard R, Rue E and Reeves R

    Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

    MRC OX-2 is a rat type I membrane glycoprotein and a member of the immunoglobulin gene superfamily that has recently been shown to be able to costimulate murine T cell proliferation (Borriello et al. J. Immunol, 158, 4548, 1997). We now report the genomic organization for the gene encoding the murine homolog of MRC OX-2 (Mox2). The gene is composed of 6 exons and 5 introns spanning a minimum of 13.7 kb. Exon 1 encodes the amino terminal four amino acids and is located in the center of a 500-bp CpG island, suggestive of the presence of a promoter. Analysis of the sequences immediately upstream of exon 1, however, do not reveal a TATA or CAAT box. We also demonstrate that in addition to the canonical transcript, composed of exons 1 through 6, this gene gives rise to an additional nonproductive transcript resulting from the absence of exon 2, which leads to a frameshift and premature translation termination. The ratio of these alternative transcripts is not regulated by mitogenic stimulation with ConA or LPS. The Mox2 gene maps to Chr 16, telomeric to the clustered T cell costimulatory molecules Cd80 and Cd86 (Reeves et al. Genomics in press).

    Funded by: NHGRI NIH HHS: HG00405; PHS HHS: A52738

    Mammalian genome : official journal of the International Mammalian Genome Society 1998;9;2;114-8

  • Prenatal differentiation of mouse vomeronasal neurones.

    Tarozzo G, Cappello P, De Andrea M, Walters E, Margolis FL, Oestreicher B and Fasolo A

    Department of Animal and Human Biology, University of Turin, Italy.

    The vomeronasal organ (VNO) subserves basic chemosensory functions in rodents, mainly related to sexual behaviour. In order to understand early stages of the VNO structural maturation, we have undertaken an immunocytochemical analysis of the VNO of fetal mice. Our results demonstrate that Olfactory Marker Protein (OMP), a marker of differentiated chemosensory cells, is already expressed in vomeronasal neurones and their fibres projecting to the accessory olfactory bulb during the last week of gestation. However, in contrast to the adult, where its expression is restricted to the medial sensory neuronal component of the VNO, during fetal development OMP is also present in cells located in the lateral non-sensory epithelial component. Some other markers of nasal chemosensory neurones, such as GAP-43/B-50, Protein Gene Product 9.5 (PGP 9.5) and carnosine are also transiently expressed in this ectopic site. These results indicate that (i) significant morphological and biochemical maturation of the VNO is achieved before birth; (ii) transient cell populations, sharing the biochemical profile of the vomeronasal chemosensory receptors, occur in ectopic areas during fetal development.

    The European journal of neuroscience 1998;10;1;392-6

  • Targeted overexpression of the neurite growth-associated protein B-50/GAP-43 in cerebellar Purkinje cells induces sprouting after axotomy but not axon regeneration into growth-permissive transplants.

    Buffo A, Holtmaat AJ, Savio T, Verbeek JS, Oberdick J, Oestreicher AB, Gispen WH, Verhaagen J, Rossi F and Strata P

    Department of Neuroscience, University of Turin, I-10125 Turin, Italy.

    B-50/GAP-43 is a nervous tissue-specific protein, the expression of which is associated with axon growth and regeneration. Its overexpression in transgenic mice produces spontaneous axonal sprouting and enhances induced remodeling in several neuron populations (; ). We examined the capacity of this protein to increase the regenerative potential of injured adult central axons, by inducing targeted B-50/GAP-43 overexpression in Purkinje cells, which normally show poor regenerative capabilities. Thus, transgenic mice were produced in which B-50/GAP-43 overexpression was driven by the Purkinje cell-specific L7 promoter. Uninjured transgenic Purkinje cells displayed normal morphology, indicating that transgene expression does not modify the normal phenotype of these neurons. By contrast, after axotomy numerous transgenic Purkinje cells exhibited profuse sprouting along the axon and at its severed end. Nevertheless, despite these growth phenomena, which never occurred in wild-type mice, the severed transgenic axons were not able to regenerate, either spontaneously or into embryonic neural or Schwann cell grafts placed into the lesion site. Finally, although only a moderate Purkinje cell loss occurred in wild-type cerebella after axotomy, a considerable number of injured transgenic neurons degenerated, but they could be partially rescued by the different transplants placed into the lesion site. Thus, B-50/GAP-43 overexpression substantially modifies Purkinje cell response to axotomy, by inducing growth processes and decreasing their resistance to injury. However, the presence of this protein is not sufficient to enable these neurons to accomplish a full program of axon regeneration.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1997;17;22;8778-91

  • The motility-associated proteins GAP-43, MARCKS, and CAP-23 share unique targeting and surface activity-inducing properties.

    Wiederkehr A, Staple J and Caroni P

    Friedrich Miescher Institute, Basel, Switzerland.

    Local regulation of the cortical cytoskeleton controls cell surface dynamics. GAP-43 and MARCKS are two abundant cytosolic protein kinase C substrates that are anchored to the cell membrane via acyl groups and interact with the cortical cytoskeleton. Each of them has been implicated in several forms of motility involving the cell surface. Although their primary sequences do not reveal significant homologies, GAP-43, MARCKS, and the cortical cytoskeleton-associated protein CAP-23 (in the following, the three proteins will be abbreviated as GMC) share a number of characteristic biochemical and biophysical properties and an unusual amino acid composition. In this study we determined whether GMC may be related functionally. In double-labeling immunocytochemistry experiments GMC accumulated at unique surface-associated structures, where they codistributed. In transfected cells GMC induced the same range of characteristic changes in cell morphology and cell surface activities, including prominent blebs and filopodia. These activities correlated with local accumulation of transgene and had characteristic features of locally elevated actin dynamics, including loss of stress fiber structures, accumulation of beta-(cytosolic) actin at cell surface protrusions, and dynamic blebbing activity. Analysis of appropriate deletion and fusion constructs revealed that the surface accumulation pattern and cell surface activities were correlated and that minimal structural requirements included acylation-mediated targeting to the cell membrane and the presence of a predominantly GMC-type sequence composition. Based on these experiments and on the results of previous studies on GAP-43, MARCKS, and CAP-23, we propose that GMC may define a class of functionally related proteins whose local accumulation promotes actin dynamics and the formation of dynamic structures at the cell periphery. Superimposed on these general properties, differences in the regulation of membrane association and binding properties of effector domains would confer individual properties to each of these proteins.

    Experimental cell research 1997;236;1;103-16

  • The costimulatory genes Cd80 and Cd86 are linked on mouse chromosome 16 and human chromosome 3.

    Reeves RH, Patch D, Sharpe AH, Borriello F, Freeman GJ, Edelhoff S and Disteche C

    Department of Physiology, 202 Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.

    Funded by: NCI NIH HHS: CA-34183, CA-40416; NHGRI NIH HHS: R01 HG00405

    Mammalian genome : official journal of the International Mammalian Genome Society 1997;8;8;581-2

  • High-resolution recombinational map of mouse chromosome 16.

    Reeves RH, Rue EE, Citron MP and Cabin DE

    Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. rreeves@welchlink.welch.jhu.edu

    Five intersubspecific backcrosses and an intercross were used to establish a sex-averaged recombinational map spanning 56 cM across most of mouse Chromosome 16 (Chr 16). A total of 123 markers were ordered using an interval mapping approach to identify 425 recombination sites in a collection of 1154 meioses from 1155 progeny generated in the six crosses. The markers include the 10 "classic" Chr 16 reference markers, 26 additional genes or transcripts including two phenotypic markers (Pit1dw and Kcnj6wv), and 87 simple sequence length polymorphisms (SSLPs). One set of monozygotic twins was detected among the 304 meioses mapped to highest resolution. The reference markers and SSLPs allow the map to be well integrated with existing maps of Chr 16. The average distance between crossover sites is less than 500 kb for most chromosomes, making this collection of recombinant chromosomes useful as a binning and ordering resource for YAC-based physical map assembly on Chr 16.

    Funded by: NHGRI NIH HHS: HG00405

    Genomics 1997;43;2;202-8

  • Disruption of local retinoid-mediated gene expression accompanies abnormal development in the mammalian olfactory pathway.

    Anchan RM, Drake DP, Haines CF, Gerwe EA and LaMantia AS

    Department of Neurobiology, Duke University Medical School, Durham, North Carolina 27710, USA.

    We have evaluated the role of retinoid signaling in the early development of the olfactory epithelium and olfactory bulb. When retinoid-mediated gene expression is blocked briefly in mouse embryos at midgestation with citral (a general alcohol dehydrogenase antagonist that is thought to interfere with retinoid synthesis), the spectrum of morphogenetic abnormalities includes disruption of olfactory pathway development. It is difficult, however, to assess the specificity of this pharmacological manipulation, insofar as it also compromises several other aspects of central nervous system development. In homozygous Pax6 mutant mice (small eye: Pax6(Sey-Neu)), there is a more discrete lesion to the olfactory pathway: The epithelium and bulb cannot be recognized at any time during development, whereas other forebrain subdivisions can still be recognized. This loss of the entire primary olfactory pathway is accompanied by a failure of retinoid-mediated gene expression limited to the frontonasal region and forebrain. Retinoid receptors are expressed in the forebrain of Pax6(Sey-Neu)/Pax6(Sey-Neu) embryos, and the mutant forebrain remains responsive to exogenous retinoic acid. However, in Pax6(Sey-Neu)/ Pax6(Sey-Neu) embryos, retinoic acid (RA) is not produced by the frontonasal mesenchyme, which normally provides local retinoid signals to the placode and forebrain. Together, these results suggest that local retinoid signaling is essential for the normal development of the mammalian olfactory pathway.

    Funded by: NICHD NIH HHS: HD 29178; NINDS NIH HHS: NS 07370

    The Journal of comparative neurology 1997;379;2;171-84

  • Analysis of the role of calmodulin binding and sequestration in neuromodulin (GAP-43) function.

    Gamby C, Waage MC, Allen RG and Baizer L

    R. S. Dow Neurological Sciences Institute, Good Samaritan Hospital and Medical Center, Portland, Oregon 97209, USA.

    We demonstrated previously that forced expression of the neuronal phosphoprotein neuromodulin (also known as GAP-43, F1, B-50, and p57) in mouse anterior pituitary AtT-20 cells enhances depolarization-mediated secretion and alters cellular morphology. Here we analyze the role of calmodulin binding by neuromodulin in these responses. In cells expressing wild-type neuromodulin, a complex with calmodulin that is sensitive to intracellular calcium and phosphorylation is localized to the plasma membrane. Transfection of several mutant forms of neuromodulin shows that the effects of this protein on secretion are dependent on both calmodulin binding and association with the plasma membrane. In contrast, the morphological changes depend only on membrane association. Thus, the multitude of effects of neuromodulin noted in previous studies may result from divergent properties of this protein.

    Funded by: NINDS NIH HHS: NS26806

    The Journal of biological chemistry 1996;271;43;26698-705

  • Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation.

    Burmeister M, Novak J, Liang MY, Basu S, Ploder L, Hawes NL, Vidgen D, Hoover F, Goldman D, Kalnins VI, Roderick TH, Taylor BA, Hankin MH and McInnes RR

    Mental Health Research Institute, University of Michigan, Ann Arbor 48109-0720, USA.

    Ocular retardation (or) is a murine eye mutation causing microphthalmia, a thin hypocellular retina and optic nerve aplasia. Here we show that mice carrying the OrJ allele have a premature stop codon in the homeobox of the Chx10 gene, a gene expressed at high levels in uncommitted retinal progenitor cells and mature bipolar cells. No CHX10 protein was detectable in the retinal neuroepithelium of orJ homozygotes. The loss of CHX10 leads both to reduced proliferation of retinal progenitors and to a specific absence of differentiated bipolar cells. Other major retinal cell types were present and correctly positioned in the mutant retina, although rod outer segments were short and retinal lamination was incomplete. These results indicate that Chx10 is an essential component in the network of genes required for the development of the mammalian eye, with profound effects on retinal progenitor proliferation and bipolar cell specification or differentiation. off

    Funded by: NCI NIH HHS: CA33093; NIGMS NIH HHS: GM18684; NINDS NIH HHS: NS26777; ...

    Nature genetics 1996;12;4;376-84

  • Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular fiber growth.

    Holtmaat AJ, Dijkhuizen PA, Oestreicher AB, Romijn HJ, Van der Lugt NM, Berns A, Margolis FL, Gispen WH and Verhaagen J

    Netherlands Institute for Brain Research, Amsterdam-ZO, The Netherlands.

    B-50/GAP-43, a neural growth-associated phosphoprotein, is thought to play a role in neuronal plasticity and nerve fiber formation since it is expressed at high levels in developing and regenerating neurons and in growth cones. Using a construct containing the coding sequence of B-50/GAP-43 under the control of regulatory elements of the olfactory marker protein (OMP) gene, transgenic mice were generated to study the effect of directed expression of B-50/GAP-43 in a class of neurons that does not normally express B-50/GAP-43, namely, mature OMP-positive olfactory neurons. Olfactory neurons have a limited lifespan and are replaced throughout adulthood by new neurons that migrate into the upper compartment of the epithelium following their formation from stem cells in the basal portion of this neuroepithelium. Thus, the primary olfactory pathway is exquisitely suited to examine a role of B-50/GAP-43 in neuronal migration, lifespan, and nerve fiber growth. We find that B-50/GAP-43 expression in adult olfactory neurons results in numerous primary olfactory axons with enlarged endings preferentially located at the rim of individual glomeruli. Furthermore, ectopic olfactory nerve fibers in between the juxtaglomerular neurons or in close approximation to blood vessels were frequently observed. This suggests that expression of B-50/GAP-43 in mature olfactory neurons alters their response to signals in the bulb. Other parameters examined, that is, migration and lifespan of olfactory neurons are normal in B-50/GAP-43 transgenic mice. These observations provide direct in vivo evidence for a role of B-50/GAP-43 in nerve fiber formation and in the determination of the morphology of axons.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1995;15;12;7953-65

  • Mapping of the calcium-sensing receptor gene (CASR) to human chromosome 3q13.3-21 by fluorescence in situ hybridization, and localization to rat chromosome 11 and mouse chromosome 16.

    Janicic N, Soliman E, Pausova Z, Seldin MF, Rivière M, Szpirer J, Szpirer C and Hendy GN

    Calcium Research Laboratory, McGill University, Montreal, Quebec, Canada.

    The calcium-sensing receptor (CASR), a member of the G-protein coupled receptor family, is expressed in both parathyroid and kidney, and aids these organs in sensing extracellular calcium levels. Inactivating mutations in the CASR gene have been described in familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT). Activating mutations in the CASR gene have been described in autosomal dominant hypoparathyroidism and familial hypocalcemia. The human CASR gene was mapped to Chromosome (Chr) 3q13.3-21 by fluorescence in situ hybridization (FISH). By somatic cell hybrid analysis, the gene was localized to human Chr 3 (hybridization to other chromosomes was not observed) and rat Chr 11. By interspecific backcross analysis, the Casr gene segregated with D16Mit4 on mouse Chr 16. These findings extend our knowledge of the synteny conservation of human Chr 3, rat Chr 11, and mouse Chr 16.

    Funded by: NHGRI NIH HHS: HG00734

    Mammalian genome : official journal of the International Mammalian Genome Society 1995;6;11;798-801

  • Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice.

    Aigner L, Arber S, Kapfhammer JP, Laux T, Schneider C, Botteri F, Brenner HR and Caroni P

    Friedrich Miescher Institute, Basel, Switzerland.

    Regulation of neurite outgrowth and structural plasticity may involve the expression of intrinsic determinants controlling growth competence. We have tested this concept by targeting constitutive expression of the growth-associated protein GAP-43 to the neurons of adult transgenic mice. Such mice showed striking spontaneous nerve sprouting at the neuromuscular junction and in the terminal field of hippocampal mossy fibers. In control mice, these nerve fibers did not express GAP-43, and did not sprout spontaneously. Lesion-induced nerve sprouting and terminal arborization during reinnervation were greatly potentiated in GAP-43-overexpressing mice. A mutant GAP-43 that cannot be phosphorylated by PKC had reduced sprout-promoting activity. The results establish GAP-43 as an intrinsic presynaptic determinant for neurite outgrowth and plasticity.

    Cell 1995;83;2;269-78

  • CHUK, a conserved helix-loop-helix ubiquitous kinase, maps to human chromosome 10 and mouse chromosome 19.

    Mock BA, Connelly MA, McBride OW, Kozak CA and Marcu KB

    Laboratory of Genetics and Biochemistry, NCI, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Helix-loop-helix proteins contain stretches of DNA that encode two amphipathic alpha-helices joined by a loop structure and are involved in protein dimerization and transcriptional regulation essential to a variety of cellular processes. CHUK, a newly described conserved helix-loop-helix ubiquitous kinase, was mapped by somatic cell hybrid analyses to human Chr 10q24-q25. Chuk and a related sequence, Chuk-rs1, were mapped to mouse chromosomes 19 and 16, respectively, by a combination of somatic cell hybrid, recombinant inbred, and backcross analyses.

    Funded by: NCI NIH HHS: CA 36246, N01-CB-21075

    Genomics 1995;27;2;348-51

  • Distinct adhesive behaviors of neurons and neural precursor cells during regional differentiation in the mammalian forebrain.

    Whitesides JG and LaMantia AS

    Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Prior to the emergence of the major functional subdivisions of the mammalian forebrain--the neocortex, hippocampus, olfactory bulb, basal ganglia, and basal forebrain--the lateral aspect of the telencephalic vesicle is distinguished by early neuronal differentiation assessed by MAP2 and GAP43 expression and increased expression of the Ca(2+)-independent/immunoglobulin superfamily cell adhesion molecules (CAMs) NCAM, L1, and TAG-1. In contrast, the ventral and medial aspects of the vesicle show little early neuronal differentiation and intermediate or undetectable levels of CAM expression. We asked whether cells from these three regions acquire distinct adhesive and recognition properties that reflect their position, state of neuronal differentiation, and level of CAM expression. In a dissociation/reaggregation assay, cells from the lateral telencephalic vesicle form the largest reaggregates while ventral reaggregates are of intermediate size and medial reaggregates are the smallest. This differential adhesion has a Ca(2+)-independent component, and cells in reaggregates from each region maintain expression of CAMs and other neuronal markers consistent with their region of origin. Furthermore, cells from the lateral telencephalon can specifically sort out from medial cells. Little adhesivity is observed prior to early neuronal differentiation and the expression of Ca(2+)-independent CAMs, when the forebrain is still a prosencephalic vesicle, nor does it follow the pattern of detectable CAM expression once forebrain rudiments are formed. Thus, cells in the early developing forebrain acquire distinct adhesive and recognition properties that reflect the concurrent emergence of regional differences in neuronal differentiation and CAM expression. These differences are transient and can only be detected in the telencephalic vesicle before and during the morphogenesis of rudiments of major forebrain subdivisions.

    Funded by: NICHD NIH HHS: HD29178

    Developmental biology 1995;169;1;229-41

  • Neuronal pathfinding is abnormal in mice lacking the neuronal growth cone protein GAP-43.

    Strittmatter SM, Fankhauser C, Huang PL, Mashimo H and Fishman MC

    Developmental Biology Laboratory, Harvard Medical School, Massachusetts General Hospital-East, Charlestown 02129.

    GAP-43 has been termed a "growth" or "plasticity" protein because it is expressed at high levels in neuronal growth cones during development and during axonal regeneration. By homologous recombination, we generated mice lacking GAP-43. The mice die in the early postnatal period. GAP-43-deficient retinal axons remain trapped in the chiasm for 6 days, unable to navigate past this midline decision point. Over the subsequent weeks of life, most GAP-43-deficient axons do enter the appropriate tracts, and the adult CNS is grossly normal. There is no evidence for interference with nerve growth rate, and cultured neurons extend neurites and growth cones in a fashion indistinguishable from controls. Thus, the GAP-43 protein is not essential for axonal outgrowth or growth cone formation per se, but is required at certain decision points, such as the optic chiasm. This is compatible with the hypothesis that GAP-43 serves to amplify pathfinding signals from the growth cone.

    Cell 1995;80;3;445-52

  • G(O), a guanine nucleotide binding protein, is expressed during neurite extension in the embryonic mouse.

    Schmidt CJ, Zubiaur M, Valenzuela D, Neer EJ and Dräger UC

    Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115.

    The developmental pattern of expression of the G protein alpha o subunit and GAP43 were compared by immunohistochemical staining of mouse embryos. Staining for alpha o and GAP43 was identical and detected throughout the developing nervous system, and the antigens first appeared in neurons at the beginning of neuronal differentiation. GAP43 and alpha o were not detected in regions containing only neuroblasts. These observations suggest that alpha o and GAP43 may not be required for the decision to pass from neuroblast to differentiated neuron, but may play a role in signal transduction during early neuronal development.

    Funded by: NEI NIH HHS: EY01938; NIGMS NIH HHS: GM36295, GM46370

    Journal of neuroscience research 1994;38;2;182-7

  • Interspecific backcrosses provide an important new tool for centromere mapping of mouse chromosomes.

    Ceci JD, Matsuda Y, Grubber JM, Jenkins NA, Copeland NG and Chapman VM

    Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Maryland 21702.

    Centromere mapping of mouse chromosomes has been problematic due to a paucity of appropriate markers. As a result, the mapping of centromeres has most often relied on the use of Robertsonian chromosomes to mark chromosome ends. Many Robertsonian translocations have been shown to suppress recombination in pericentric regions; therefore, centromere mapping data generated by using Robertsonian chromosomes must be interpreted with caution. We have utilized a new tool for centromere mapping that is applicable to all mouse chromosomes (except the Y chromosome) and that potentially overcomes the inherent limitations of using Robertsonian translocations. Briefly, an interspecific backcross mapping panel was constructed from crosses of C57BL/6Ros and Mus spretus mice. The centromere of each chromosome was subsequently typed by in situ hybridization, using a major satellite probe that uniformly labels C57BL/6Ros centromeres but hybridizes only weakly to M. spretus centromeres. Genetic markers that were already known to map in the proximal region of each of the mouse chromosomes were then typed by segregation analyses of restriction fragment length polymorphisms. These studies have made it possible to align the interspecific genetic map of each of the mouse autosomes and the X chromosome with respect to the centromere. They also provide a basis for comparison with centromere mapping data generated previously by other means.

    Funded by: NCI NIH HHS: N01-CO-74101; NHGRI NIH HHS: HG-00170; NIGMS NIH HHS: GM-33160

    Genomics 1994;19;3;515-24

  • Molecular characterization and mapping of murine genes encoding three members of the stefin family of cysteine proteinase inhibitors.

    Tsui FW, Tsui HW, Mok S, Mlinaric I, Copeland NG, Gilbert DJ, Jenkins NA and Siminovitch KA

    Department of Medicine, University of Toronto, Ontario, Canada.

    Stefins or Type 1 cystatins belong to a large, evolutionarily conserved protein superfamily, the members of which inhibit the papain-like cysteine proteinases. We report here on the molecular cloning and chromosomal localization of three newly identified members of the murine stefin gene family. These genes, designated herein as mouse stefins 1, 2, and 3, were isolated on the basis of their relatively increased expression in moth-eaten viable compared to normal congenic mouse bone marrow cells. The open reading frames of the stefin cDNAs encode proteins of approximately 11.5 kDa that show between 50 and 92% identity to sequences of stefins isolated from various other species. Data from Southern analysis suggest that the murine stefin gene family encompasses at least 6 and possibly 10-20 members, all of which appear to be clustered in the genome. Analysis of interspecific backcross mice indicates that the genes encoding the three mouse stefins all map to mouse chromosome 16, a localization that is consistent with the recent assignment of the human stefin A gene to a region of conserved homology between human chromosome 3q and the proximal region of mouse chromosome 16.

    Funded by: PHS HHS: R01-C0-74101

    Genomics 1993;15;3;507-14

  • The positions of 12 simple sequence repeat markers relative to reference loci on mouse chromosome 16.

    Irving NG, Citron MP and Reeves RH

    Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

    The genetic map positions of 12 simple sequence repeat (SSR) markers spanning mouse Chromosome (Chr) 16 were determined relative to reference markers on that chromosome. Interval mapping data were obtained with a panel of DNAs from two intersubspecific backcrosses. All but one of the markers were typed by us of nonradioactive polymerase chain reaction (PCR) products analyzed on agarose gels. The marker order was determined to be Prm-1, D16Mit9, Igl-1, D16Mit29, D16Mit1/D16Mit2, Smst, D16Mit4, D16Mit11, Gap43, D16Mit14, D16Mit30, D16Mit5, Pit-1, D16Mit27, D16H21S16 (formerly D21S16h), D16Mit19, App, D16Mit7, Sod-1. Two of these markers mapped to the known human Chr 21 (HSA21)/Chr 16 conserved linkage group. Nine additional SSR markers could not be typed because they were not polymorphic (four markers), did not amplify MOLD/Rk DNA (three markers), or failed to give PCR products under a range of conditions (two markers). A subset of the most robust SSRs provide a useful marker set for the analysis of previously unmapped crosses.

    Funded by: NHGRI NIH HHS: HG00405

    Mammalian genome : official journal of the International Mammalian Genome Society 1993;4;7;364-7

  • Transcriptional regulation of neuromodulin (GAP-43) in mouse neuroblastoma clone N1E-115 as evaluated by the RT/PCR method.

    Robbins M and McKinney M

    Mayo Clinic Jacksonville, FL 32224.

    The steady-state level of the neuromodulin transcript in the neuron-like N1E-115 cell line was measured with a method combining reverse transcription and the polymerase chain reaction (RT/PCR). Total RNA was isolated from N1E-115 cells and treated with DNAse to remove residual DNA; cDNA was synthesized from this RNA by priming with random hexamers. For PCR amplification, primers for neuromodulin were designed for regions of the coding sequence that were identical in mouse, rat, and human. In one approach (the 'ratio method'), variations in RNA yield and cDNA synthesis efficiency were controlled for by amplifying a reference (housekeeping) gene (glyceraldehyde phosphate dehydrogenase; GAPDH). To control for inter-experimental variations in PCR amplification efficiencies the data were analyzed on semi-logarithmic plots, with which the relative levels of the starting templates could be determined by extrapolating the plots to cycle number zero (0). In another approach with RT/PCR (the 'spiking method'), the absolute level of N1E-115 neuromodulin cDNA was assessed by adding known amounts of cloned human neuromodulin template to the RT/PCR assay of N1E-115 nucleic acid and comparing the increased yield of product across cycles. When the spike was added at either the cDNA level (in the form of double-stranded DNA) or at the total RNA level (as sense RNA), the levels of N1E-115 calculated were virtually the same: 509 fg and 495 fg of coding region per ug total RNA in confluent N1E-115 cells, respectively. Treatment of N1E-115 cells with 2% dimethylsulfoxide for three days elevated neuromodulin mRNA levels 5.6-fold. Conversely, treatment of N1E-115 cells with 100 nM phorbol myristate acetate for 24 h decreased the level of neuromodulin mRNA by 70%. Under carefully controlled conditions and within certain limits of precision, the RT/PCR method appears to be suitable for assessing the level of low abundance mRNA under various pharmacologically-induced conditions.

    Funded by: NIA NIH HHS: AG09973

    Brain research. Molecular brain research 1992;13;1-2;83-92

  • Genetic mapping of two DNA markers, D16Ros1 and D16Ros2, flanking the mutation site in the chakragati mouse, a transgenic insertional mutant.

    Ratty AK, Matsuda Y, Elliott RW, Chapman VM and Gross KW

    Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263.

    We present here the genetic mapping of two novel loci, D16Ros1 and D16Ros2, to mouse Chromosome (Chr) 16. The probes for these loci were genomic fragments isolated from the chakragati mouse, a behavioral mutant resulting from insertional mutagenesis during the course of making transgenic mice. D16Ros1 and D16Ros2 were first mapped by recombinant inbred (RI) strain analysis and subsequently by the analysis of 145 progeny of two interspecific backcrosses between Mus domesticus and Mus spretus. These progeny had been typed for the centromere and this allowed mapping of D16Ros1 and D16Ros2 relative to the centromere. The other markers included in this study were Prm-1, Gap43 and Sod-1. The genetic map generated spanned 47.5 cM from the centromere to Sod-1, the most distal marker mapped here. The linkage data presented here should prove useful in mapping other loci relative to the centromere of Chr 16.

    Funded by: NIGMS NIH HHS: GM28464/HG00342, GM30248, GM33160

    Mammalian genome : official journal of the International Mammalian Genome Society 1992;3;1;5-10

  • Mapping HSA 3 loci in cattle: additional support for the ancestral synteny of HSA 3 and 21.

    Threadgill DS and Womack JE

    Department of Veterinary Pathology, Texas A&M University, College Station 77843.

    Homologs to genes residing on human chromosome 3 (HSA 3) map to four mouse chromosomes (MMU) 3, 6, 9, and 16. In the bovine, two syntenic groups that contain HSA 3 homologs, unassigned syntenic groups 10 (U10) and 12 (U12), have been defined. U10 also contains HSA 21 genes, which is similar to the situation seen on MMU 16, whereas U12 apparently contains only HSA 3 homologs. The syntenic arrangement of other HSA 3 homologs in the bovine was investigated by physically mapping five genes through segregation analysis of a bovine-hamster hybrid somatic cell panel. The genes mapped include Friend-murine leukemia virus integration site 3 homolog (FIM3; HSA 3/MMU 3), sucrase-isomaltase (SI) and glutathione peroxidase 1 (GPX1) (HSA 3/MMU ?), murine leukemia viral (v-raf-1) oncogene homolog 1 (RAF1; HSA 3/MMU 6), and ceruloplasmin (CP; HSA 3/MMU 9). FIM3, SI, and CP mapped to bovine syntenic group U10, while RAF1 and GPX1 mapped to U12.

    Genomics 1991;11;4;1143-8

  • Mouse chromosome-specific markers generated by PCR and their mapping through interspecific backcrosses.

    Irving NG and Brown SD

    Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, London, United Kingdom.

    We have utilized an oligonucleotide primer from the 3' end of the mouse L1 repeat element for amplification of mouse-specific inter-repeat PCR products from Chinese hamster/mouse somatic cell hybrids. PCR of a Chinese hamster/mouse somatic cell hybrid (96AZ2), containing only mouse chromosome 16, produced a range of mouse-specific bands. Two of the mouse-specific PCR products, of 250 and 580 bp, have been confirmed as originating from mouse chromosome 16 by somatic cell hybrid analysis. Both the 250- and 580-bp PCR products have been sequenced and demonstrate the expected sequence organization. Furthermore, both the 250- and 580-bp markers have been genetically mapped in detail to mouse chromosome 16 by direct hybridization to inter-repeat PCR products of progeny DNAs from Mus domesticus/Mus spretus interspecific backcrosses.

    Genomics 1991;11;3;679-86

  • Characterization of the opposite-strand genes from the mouse bidirectionally transcribed HTF9 locus.

    Bressan A, Somma MP, Lewis J, Santolamazza C, Copeland NG, Gilbert DJ, Jenkins NA and Lavia P

    Dipartimento di Genetica e Biologia Molecolare, Università La Sapienza, Rome, Italy.

    The mouse HTF9 locus contains two genes that are bidirectionally transcribed with opposite polarity from a shared CpG-rich island. Both genes were previously shown to be expressed in a housekeeping fashion in mouse. We have now determined the molecular organization of the genes over 12 kb surrounding the island. In addition, we show that the HTF9 locus resides in the proximal region of mouse chromosome 16. We have sequenced the cDNAs corresponding to both divergent transcripts. Both genes appear to code for novel proteins that are structurally unrelated to each other. Finally, we show that both genes are highly conserved and efficiently expressed in human cells.

    Funded by: NCI NIH HHS: N01-CO-74101

    Gene 1991;103;2;201-9

  • The multipoint genetic mapping of mouse chromosome 16.

    Irving NG, Hardy JA and Brown SD

    Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, London, United Kingdom.

    Utilizing a Mus spretus/Mus domesticus (C57BL/10) interspecific backcross, we have constructed a multipoint genetic map of mouse chromosome 16 that extends 43.2 cM from the proximal Prm-1 locus to the distal Ets-2 locus. The genetic map incorporates three new markers: D16Smh6, a random genomic clone; Pgk-1ps1, a phosphoglycerate kinase pseudogene; and the growth-associated protein Gap43. The map position of Gap43 indicates the presence, on mouse chromosome 16, of a significant-size conserved linkage group with human chromosome 3.

    Genomics 1991;9;2;386-9

  • Comparison of interspecific to intersubspecific backcrosses demonstrates species and sex differences in recombination frequency on mouse chromosome 16.

    Reeves RH, Crowley MR, Moseley WS and Seldin MF

    Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

    One hundred fourteen progeny from an interspecific backcross between laboratory mice and M. spretus were typed for six markers spanning most of mouse Chromosome (Chr) 16. Additional maps of 9-10 markers of this chromosome were derived from analysis of over 500 progeny from four backcrosses between inbred laboratory strains and members of the Mus musculus group, M.m. musculus and M.m. molossinus (subspecies). The results of these analyses confirmed the gene order: (CEN)-Prm-1/Prm-2-Igl-1-Smst-Mtv-6-Gap43-Pit-1(dw)- D21S16h-App-Sod-1-Ets-2-Mx. Maps produced from these five crosses were of similar lengths, but recombination in several regions was affected by sex of the F1 parent or by the combination of strains used in the cross. As reported previously, recombination frequencies were elevated significantly at the distal end of the chromosome in a cross using F1 males. The male map showed significant compression in the interval Smst to Gap43. Both male and female intersubspecific maps were expanded near the proximal and distal ends of the chromosome relative to the interspecific cross. The spretus cross was compressed in the proximal interval, Prm-1-Igl-1-Smst, and was slightly expanded in the Smst-Gap43 interval, relative to intersubspecific crosses using F1 females. Female intersubspecific maps were expanded about 50% near the distal end of the chromosome when compared to the interspecific cross. The expansion or compression of maps using different strain or sex combinations has implications for the efficient production of high resolution recombinational maps of the mouse genome.

    Funded by: NHGRI NIH HHS: HG00101; NICHD NIH HHS: HD22262

    Mammalian genome : official journal of the International Mammalian Genome Society 1991;1;3;158-64

  • The Pit-1 transcription factor gene is a candidate for the murine Snell dwarf mutation.

    Camper SA, Saunders TL, Katz RW and Reeves RH

    Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109-0618.

    Two nonallelic mouse mutations with severe dwarf phenotypes are characterized by a lack of growth hormone, prolactin, and thyroid stimulating hormone. The cells that normally synthesize these pituitary hormones express a common transcription factor called GHF-1 or Pit-1. Using an intersubspecific backcross, we have demonstrated tight linkage of the Pit-1 and Snell dwarf (dw) genes on mouse chromosome 16. No recombination was observed between Pit-1 and dw in 110 individuals examined. Southern blot analysis of genomic DNA reveals that the Pit-1 gene is rearranged in C3H/HeJ-dwJ/dw mice but not in coisogenic +/+ animals, providing molecular evidence that a lesion in the Pit-1 gene results in the Snell dwarf phenotype. Demonstration of low levels of Pit-1 expression in Ames dwarf (df) mice implies that both Pit-1 and df expression may be required for pituitary differentiation.

    Funded by: NICHD NIH HHS: HD22262; NIGMS NIH HHS: T32-GM07315

    Genomics 1990;8;3;586-90

  • Sex, strain, and species differences affect recombination across an evolutionarily conserved segment of mouse chromosome 16.

    Reeves RH, Crowley MR, O'Hara BF and Gearhart JD

    Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196.

    A region of substantial genetic homology exists between human chromosome 21 (HSA21) and mouse chromosome 16 (MMU16). Analysis of 520 backcross animals has been used to establish gene order in the homologous segment. D21S16h and Mx are shown to represent the known proximal and distal limits of homology between the chromosomes, while Gap43, whose human homolog is on HSA3, is the next proximal marker on MMU16 that has been mapped in the human genome. Recombination frequencies (RFs) in four intervals defined by five loci in the HSA21-homologous region of MMU16 were analyzed in up to 895 progeny of eight different backcrosses. Two of the eight crosses were made with F1 males and six with F1 females. The average RF of 0.249 in 265 backcross progeny of F1 males was significantly higher than the 0.106 average recombination in 320 progeny of F1 females in the interval from D21S16h to Ets-2. This is in contrast to HSA21, which shows higher RFs in female meiosis in the corresponding region. Considerable variation in RF was observed between crosses involving different strains, both in absolute and in relative sizes of the intervals measured. The highest RFs occurred in a cross between the laboratory strain C57BL/6 and MOLD/Rk, an inbred strain derived from Mus musculus molossinus. RFs on this cross were nearly fivefold higher than those reported previously for an interspecific cross between C57BL/6 and Mus spretus.

    Funded by: NICHD NIH HHS: HD22262, HD24605

    Genomics 1990;8;1;141-8

  • B-50/GAP43 Expression Correlates with Process Outgrowth in the Embryonic Mouse Nervous System.

    Biffo S, Verhaagen J, Schrama LH, Schotman P, Danho W and Margolis FL

    Department of Neurosciences, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110, USA.

    The hypothesis that B-50/GAP43, a membrane-associated phosphoprotein, is involved in process outgrowth has been tested by studying the developmental pattern of expression of B-50/GAP43 mRNA and protein during mouse neuroembryogenesis. B-50/GAP43 mRNA is first detectable at embryonic day 8.5 (E8.5) in the presumptive acoustico-facialis ganglion. Subsequently, both B-50/GAP43 mRNA and protein were co-expressed in a series of neural structures: in the ventral neural tube (from E9.5) and dorsal root ganglia (from E10.5), in the marginal layer of the neuroepithelium surrounding the brain vesicles and in the cranial ganglia (from E9.5), in the autonomic nervous system (from E10.5), in the olfactory neuroepithelium and in the mesenteric nervous system (from E11.5), in a continuum of brain regions (from E12.5) and in the retina (from E13.5). Immunoreactive fibers were always seen arising from these regions when they expressed B-50/GAP43 mRNA. The spatial and temporal pattern of B-50/GAP43 expression demonstrates that this protein is absent from neuroblasts and consistently appears in neurons committed to fiber outgrowth. The expression of the protein in immature neurons is independent of their embryological origin. Our detailed study of B-50/GAP43 expression during mouse neuroembryogenesis supports the view that this protein is involved in a process common to all neurons elaborating fibers.

    The European journal of neuroscience 1990;2;6;487-499

  • Genetic mapping of the Mx influenza virus resistance gene within the region of mouse chromosome 16 that is homologous to human chromosome 21.

    Reeves RH, O'Hara BF, Pavan WJ, Gearhart JD and Haller O

    Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

    A total of 318 progeny from four backcrosses involving different laboratory strains and subspecies of Mus musculus were analyzed to map the Mx gene to the region of mouse chromosome 16 (MMU 16) which is homologous to human chromosome 21 (HSA 21). This result suggests that Mx will be found in the region of HSA 21 which has been implicated in Down syndrome when inherited in three copies.

    Funded by: NICHD NIH HHS: P01 HD 19920, R01 HD 22262

    Journal of virology 1988;62;11;4372-5

  • Human GAP-43: its deduced amino acid sequence and chromosomal localization in mouse and human.

    Kosik KS, Orecchio LD, Bruns GA, Benowitz LI, MacDonald GP, Cox DR and Neve RL

    Department of Neurology (Neuroscience), Harvard Medical School, Boston, Massachusetts 02115.

    The growth-associated protein (GAP-43) is considered a crucial component of an effective regenerative response in the nervous system. Its phosphorylation by protein kinase C correlates with long-term potentiation. Sequence analysis of human cDNAs coding for this protein shows that the human GAP-43 gene is highly homologous to the rat gene; this homology extends into the 3'-untranslated region. However, the human protein contains a 10 amino acid insert. Somatic cell hybrids demonstrate localization of the GAP-43 gene to human chromosome 3 and to mouse chromosome 16.

    Funded by: NICHD NIH HHS: HD18658; NINDS NIH HHS: NS00835, NS25830; ...

    Neuron 1988;1;2;127-32

  • Characterization of murine cDNAs encoding P-57, a neural-specific calmodulin-binding protein.

    Cimler BM, Giebelhaus DH, Wakim BT, Storm DR and Moon RT

    Polyclonal antibodies raised against bovine brain P-57, a neural-specific calmodulin-binding protein, were used to isolate murine cDNAs encoding P-57 from murine brain cDNA libraries in the expression vector lambda gt 11. Two of the overlapping clones contained an open reading frame encoding a polypeptide of 227 amino acid residues (predicted Mr, 23,635), a 163-nucleotide 5'-untranslated sequence, and a 403-nucleotide 3'-untranslated sequence. Hydrophobicity analysis of the predicted polypeptide indicated the lack of any considerable stretch of hydrophobic residues that may span the membrane. This is consistent with prior data suggesting that P-57 exists in a soluble, as well as a membrane-associated, form. The predicted amino acid composition of P-57 is rather unusual in that it is highly enriched in alanine, glutamic acid, and lysine residues, and relatively enriched with proline residues. This amino acid composition accounts for the very low helical content of the predicted polypeptide. A search of the GenBank and EMBL sequence data banks (GenBank Inc., release 44.0 (August, 1986); European Molecular Biology Library, release 8.0 (April, 1986] indicated that the P-57 nucleotide sequence shows no significant homology to any reported sequences. RNA blot analysis of brain, heart, liver, and testes RNA revealed that cDNAs detect P-57 transcripts of 1.5 kilobases in brain, but not in other tissues. Genome blot analysis was consistent with P-57 being encoded by a single or small number of genes. These data demonstrate that the accumulation of this novel calmodulin-binding polypeptide in neural tissue is controlled primarily at the level of RNA abundance.

    Funded by: NIGMS NIH HHS: 5 T32 GM07270, GM33708

    The Journal of biological chemistry 1987;262;25;12158-63

  • Genetic mapping of Prm-1, Igl-1, Smst, Mtv-6, Sod-1, and Ets-2 and localization of the Down syndrome region on mouse chromosome 16.

    Reeves RH, Gallahan D, O'Hara BF, Callahan R and Gearhart JD

    Molecular probes were used as markers in the backcross (Czech II X BALB/cPt) X Czech II to determine the positions of six genes on mouse chromosome 16 (MMU 16). The order of the genes mapped is (centromere), protamine-1 (Prm-1), immunoglobulin lambda 1 light chain (Igl-1), preprosomatostatin (Smst), an endogenous mouse mammary tumor virus locus (Mtv-6), and two more distal sequences, superoxide dismutase, cytoplasmic form (Sod-1), and the proto-oncogene sequence Ets-2. The largest recombination frequency between any two adjacent markers is 24 cM, and thus the position of any marker on MMU 16 that is polymorphic between these two strains can be readily determined in this backcross. A region of MMU 16 which corresponds to the Down syndrome region of human chromosome 21 is located near the distal end of the chromosome.

    Funded by: PHS HHS: P01-19920

    Cytogenetics and cell genetics 1987;44;2-3;76-81

Gene lists (10)

Gene List Source Species Name Description Gene count
L00000001 G2C Mus musculus Mouse PSD Mouse PSD adapted from Collins et al (2006) 1080
L00000003 G2C Mus musculus Mouse clathrin Mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000004 G2C Mus musculus Mouse Synaptosome Mouse Synaptosome adapted from Collins et al (2006) 152
L00000007 G2C Mus musculus Mouse NRC Mouse NRC adapted from Collins et al (2006) 186
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
L00000019 G2C Mus musculus Pocklington M1 Cluster 1 (mouse) from Pocklington et al (2006) 21
L00000060 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus (ortho) 748
L00000062 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus 984
L00000070 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list (ortho) 1461
L00000072 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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